Modification of fatty acid metabolism in plants

ABSTRACT

Methods and systems to modify fatty acid biosynthesis and oxidation in plants to make new polymers are provided. Two enzymes are essential: a hydratase such as D-specific enoyl-CoA hydratase, for example, the hydratase obtained from  Aeromonas caviae , and a β-oxidation enzyme system. Some plants have a β-oxidation enzyme system which is sufficient to modify polymer synthesis when the plants are engineered to express the hydratase. Examples demonstrate production of polymer by expression of these enzymes in transgenic plants. Examples also demonstrate that modifications in fatty acid biosynthesis can be used to alter plant phenotypes, decreasing or eliminating seed production and increasing green plant biomass, as well as producing polyhydroxyalkanoates.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Priority is claimed to U.S. application Ser. No. 09/263,406,filed Mar. 5, 1999, which claims priority to U.S. Provisionalapplication Serial No. 60/077,107, filed Mar. 6, 1998.

BACKGROUND OF THE INVENTION

[0002] The present invention is generally in the field of transgenicplant systems for the production of polyhydroxyalkanoate materials,modification of triglycerides and fatty acids, and methods for alteringseed production in plants.

[0003] Methods for producing stable transgenic plants for agronomiccrops have been developed over the last 15 years. Crops have beengenetically modified for improvements in both input and output traits.In the former traits, tolerance to specific agrochemicals has beenengineered into crops, and specific natural pesticides, such as theBacillus thuringenesis toxin, have been expressed directly in the plant.There also has been significant progress in developing male sterilitysystems for the production of hybrid plants. With respect to outputtraits, crops are being modified to increase the value of the product,generally the seed, grain, or fiber of the plant. Critical metabolictargets include the modification of starch, fatty acid, and oilbiosynthetic pathways.

[0004] There is considerable commercial interest in producing microbialpolyhydroxyalkanoate (PHA) biopolymers in plant crops. See, for example,U.S. Pat. Nos. 5,245,023 and 5,250,430 to Peoples and Sinskey; U.S. Pat.No. 5,502,273 to Bright et al.; U.S. Pat. No. 5,534,432 to Peoples andSinskey; U.S. Pat. No. 5,602,321 to John; U.S. Pat. No. 5,610,041 toSomerville et al.; PCT WO 91/00917; PCT WO 92/19747; PCT WO 93/02187;PCT WO 93/02194; PCT WO 94/12014; Poirier et al., Science 256:520-23(1992); van der Leij & Witholt, Can. J. Microbiol. 41(supplement):222-38(1995); Nawrath & Poirier, The International Symposium on BacterialPolyhydroxyalkanoates, (Eggink et al., eds.) Davos Switzerland (Aug.18-23, 1996); Williams and Peoples, CHEMTECH 26: 38-44 (1996), and therecent excellent review by Madison, L. and G. Husiman, Microbiol. Mol.Biol. 21-53 (March 1999). PHAs are natural, thermoplastic polyesters andcan be processed by traditional polymer techniques for use in anenormous variety of applications, including consumer packaging,disposable diaper linings and garbage bags, food and medical products.

[0005] Early studies on the production of polyhydroxybutyrate in thechloroplasts of the experimental plant system Arabidopsis thalianaresulted in the accumulation of up to 14% of the leaf dry weight as PHB(Nawrath et al., 1993). Arabidopsis, however, has no agronomic value.Moreover, in order to economically produce PHAs in agronomic crops, itis desirable to produce the PHAs in the seeds, so that the currentinfrastructure for harvesting and processing seeds can be utilized. Theoptions for recovery of the PHAs from plant seeds (PCT WO 97/15681) andthe end use applications (Williams & Peoples, CHEMTECH 26:38-44 (1996))are significantly affected by the polymer composition. Therefore, itwould be advantageous to develop transgenic plant systems that producePHA polymers having a well-defined composition, as well as produce PHApolymer in specific locations within the plants and/or seeds.

[0006] Careful selection of the PHA biosynthetic enzymes on the basis oftheir substrate specificity allows for the production of PHA polymers ofdefined composition in transgenic systems (U.S. Pat. Nos. 5,229,279;5,245,023; 5,250,430; 5,480,794; 5,512,669; 5,534,432; 5,661,026; and5,663,063).

[0007] In bacteria, each PHA group is produced by a specific pathway. Inthe case of the short pendant group PHAs, three enzymes are involved:β-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase. Thehomopolymer PHB, for example, is produced by the condensation of twomolecules of acetyl-coenzyme A to give acetoacetyl-coenzyme A. Thelatter then is reduced to the chiral intermediateR-3-hydroxybutyryl-coenzyme A by the reductase, and subsequentlypolymerized by the PHA synthase enzyme. The PHA synthase notably has arelatively wide substrate specificity which allows it to polymerizeC3-C5 hydroxy acid monomers including both 4-hydroxy and 5-hydroxy acidunits. This biosynthetic pathway is found in a number of bacteria suchas Alcaligenes eutrophus, A. latus, Azotobacter vinlandii, and Zoogloearamigera. Long pendant group PHAs are produced for example by manydifferent Pseudomonas bacteria. Their biosynthesis involves theβ-oxidation of fatty acids and fatty acid synthesis as routes to thehydroxyacyl-coenzyme A monomeric units. The latter then are converted byPHA synthases which have substrate specificities favoring the largerC6-C14 monomeric units (Peoples & Sinskey, 1990).

[0008] In the case of the PHB-co-HX copolymers which usually areproduced from cells grown on fatty acids, a combination of these routescan be responsible for the formation of the different monomeric units.Indeed, analysis of the DNA locus encoding the PHA synthase gene inAeromonas caviae, which produces the copolymerPHB-co-3-hydroxyhexanoate, was used to identify a gene encoding aD-specific enoyl-CoA hydratase responsible for the production of theD-β-hydroxybutyryl-CoA and D-β-hydroxyhexanoyl-CoA units (Fukui & Doi,J. Bacteriol. 179:4821-30 (1997); Fukui et. al., J. Bacteriol.180:667-73 (1998)). Other sources of such hydratase genes and enzymesinclude Alcaligenes, Pseudomonas, and Rhodospirillum.

[0009] The enzymes PHA synthase, acetoacetyl-CoA reductase, andβ-ketothiolase, which produce the short pendant group PHAs in A.eutrophus, are coded by an operon comprising the phbC-phbA-phbB genes;Peoples et al., 1987; Peoples & Sinskey, 1989). In the Pseudomonasorganisms, the PHA synthases responsible for production of the longpendant group PHAs have been found to be encoded on the pha locus,specifically by thephaA and phaC genes (U.S. Pat. Nos. 5,245,023 and5,250,430; Huisman et. al., J. Biol. Chem. 266:2191-98 (1991)). Sincethese earlier studies, a range of PHA biosynthetic genes have beenisolated and characterized or identified from genome sequencingprojects. Known PHA biosynthetic genes include: Aeronomas caviae (Fukui& Doi, 1997, J. Bacteriol. 179:4821-30); Alcaligenes eutrophus (U.S.Pat. Nos. 5,245,023; 5,250,430; 5,512,669; and 5,661,026; Peoples &Sinskey, J. Biol. Chem. 264:15298-03 (1989)); Acinetobacter (Schembriet. al., FEMS Microbiol. Lett. 118:145-52 (1994)); Chromatium vinosum(Liebergesell & Steinbuchel, Eur. J. Biochem. 209:135-50 (1992));Methylobacterium extorquens (Valentin & Steinbuchel, Appl. Microbiol.Biotechnol. 39:309-17 (1993)); Nocardia corallina (GENBANK Accession No.AF019964; Hall et. al., 1998, Can. J. Microbiol. 44:687-69); Paracoccusdenitrificans (Ueda et al., J. Bacteriol. 178:774-79 (1996); Yabutaniet. al., FEMS Microbiol. Lett. 133:85-90 (1995)); Pseudomonas acidophila(Umeda et. al., 1998, Applied Biochemistry and Biotechnology,70-72:341-52); Pseudomonas sp. 61-3 (Matsusaki et al., 1998, J.Bacteriol. 180:6459-67); Nocardia corallina; Pseudomonas aeruginosa(Timm & Steinbuchel, Eur. J. Biochem. 209:15-30 (1992)); P. oleovorans(U.S. Pat. Nos. 5,245,023 and 5,250,430; Huisman et. al., J. Biol. Chem.266(4):2191-98 (1991); Rhizobium etli (Cevallos et. al., J. Bacteriol.178:1646-54 (1996)); R. meliloti (Tombolini et. al., Microbiology141:2553-59 (1995)); Rhodococcus ruber (Pieper-Furst & Steinbuchel, FEMSMicrobiol. Lett. 75:73-79 (1992)); Rhodospirillum rubrum (Hustede et.al., FEMS Microbiol. Lett 93:285-90 (1992)); Rhodobacter sphaeroides(Hustede et. al., FEMS Microbiol. Rev. 9:217-30 (1992); Biotechnol.Lett. 15:709-14 (1993); Synechocystis sp. (DNA Res. 3:109-36 (1996));Thiocapsiae violacea (Appl. Microbiol. Biotechnol. 38:493-501 (1993))and Zoogloea ramigera (Peoples et. al., J. Biol. Chem. 262:97-102(1987); Peoples & Sinskey, Molecular Microbiology 3:349-57 (1989)). Theavailability of these genes or their published DNA sequences shouldprovide a range of options for producing PHAs.

[0010] PHA synthases suitable for producing PHB-co-HH copolymerscomprising from 1-99% HH monomers are encoded by the Rhodococcus ruber,Rhodospirillum rubrum, Thiocapsiae violacea, and Aeromonas caviae PHAsynthase genes. PHA synthases useful for incorporating 3-hydroxyacids of6-12 carbon atoms in addition to R-3-hydroxybutyrate i.e. for producingbiological polymers equivalent to the chemically synthesized copolymersdescribed in PCT WO 95/20614, PCT WO 95/20615, and PCT WO 95/20621 havebeen identified in a number of Pseudomonas and other bacteria(Steinbüchel & Wiese, Appl. Microbiol Biotechnol. 37:691-97 (1992);Valentin et al., Appl. Microbiol. Biotechnol. 36:507-14 (1992); Valentinet al., Appl. Microbiol. Biotechnol. 40:710-16 (1994); Lee et al., AppLMicrobiol. Biotechnol. 42:901-09 (1995); Kato et al., Appl. Microbiol.Biotechnol. 45:363-70 (1996); Abe et al., Int. J. Biol. Macromol.16:115-19 (1994); Valentin et al., Appl. Microbiol. Biotechnol.46:261-67 (1996)) and can readily be isolated as described in U.S. Pat.Nos. 5,245,023 and 5,250,430. The PHA synthase from P. oleovorans (U.S.Pat. Nos. 5,245,023 and 5,250,430; Huisman et. al., J. Biol. Chem.266(4): 2191-98 (1991)) is suitable for producing the long pendant groupPHAs. Plant genes encoding β-ketothiolase also have been identified(Vollack & Bach, Plant Physiol. 111:1097-107 (1996)).

[0011] Despite this ability to modify monomer composition by selectionof the syntheses and substrates, it is desirable to modify otherfeatures of polymer biosynthesis, such as fatty acid metabolism.

[0012] It is therefore an object of the present invention to provide amethod and DNA constructs to introduce fatty acid oxidation enzymesystems for manipulating the cellular metabolism of plants.

[0013] It is another object of the present invention to provide methodsfor enhancing the production of PHAs in plants, preferably in theoilseeds thereof.

SUMMARY OF THE INVENTION

[0014] Methods and systems to modify fatty acid biosynthesis andoxidation in plants to make new polymers are described. Two enzymes areessential: a hydratase such as D-specific enoyl-CoA hydratase, forexample, the hydratase obtained from Aeromonas caviae, and a β-oxidationenzyme system. Some plants have a β-oxidation enzyme system which issufficient to modify polymer synthesis when the plants are engineered toexpress the hydratase. Tissue specific and constitutive promoters wereused to regulate and direct polymer production. Fusion constructsenhance polymer production.

[0015] Examples demonstrate production of polymer by expression of theseenzymes in transgenic plants. Examples also demonstrate thatmodifications in fatty acid biosynthesis can be used to alter plantphenotypes, decreasing or eliminating seed production and increasinggreen plant biomass, as well as producing PHAs. Use of the phaseolinpromoter can be used to induce male sterility. Tissue specific promotersin fusion constructs were used to modify production within regions ofthe seeds.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a schematic of fatty acid β-oxidation routes to producepolyhydroxyalkanoate monomers.

[0017]FIG. 2 is a schematic showing plasmid constructs pSBS2024 andpSBS2025.

[0018]FIGS. 3A and 3B are schematics showing plasmid constructs pCGmf124and pCGmf125.

[0019]FIGS. 4A and 4B are schematics showing plasmid constructs pmf1249and pmf1254.

[0020]FIGS. 5A and 5B are schematics showing plasmid constructs pCGmf224and pCGmf225.

[0021]FIGS. 6A and 6B are schematics showing plasmid constructspCGmf1P2S and pCGmf2P1S.

[0022]FIG. 7 is a schematic showing plasmid constructs pCGm1124,pCGmf125,pCGMI5028, pCGmf224, pCGmf225, pCGMI5038, pCGmf1P2S, pCGmf2P1S,pCGMI5006, pCGmf138, pCGmf1A2P, and pCGmf5034.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Methods and DNA constructs for manipulating the cellularmetabolism of plants by introducing fatty acid oxidation enzyme systemsinto the cytoplasm or plastids of developing oilseeds or green tissuethrough the use of tissue specific and/or constitutive promoters, areprovided. Fatty acid oxidation systems typically comprise several enzymeactivities including a β-ketothiolase enzyme activity which utilizes abroad range of β-ketoacyl-CoA substrates.

[0024] It surprisingly was found that expression of at least one ofthese transgenes from the bean phaseolin promoter results in malesterility. Interestingly, these plants did not set seed, but insteadproduced higher than normal levels of biomass (e.g., leafs, stems,stalks). Therefore the methods and constructs described herein also canbe used to create male sterile plants, for example, for hybridproduction or to increase the production of biomass of forage, such asalfalfa or tobacco. Plants generated using these methods and DNAconstructs are useful for producing polyhydroxyalkanoate biopolymers orfor producing novel oil compositions.

[0025] The methods described herein include the subsequent incorporationof additional transgenes, in particular encoding additional enzymesinvolved in fatty acid oxidation or polyhydroxyalkanoate biosynthesis.For polyhydroxyalkanoate biosynthesis, the methods include theincorporation of transgenes encoding enzymes, such as NADH and/or NADPHacetoacetyl-CoenzymeA reductases, PHB synthases, PHA synthases,acetoacetyl-CoA thiolase, hydroxyacyl-CoA epimerases,delta3-cis-delta2-trans enoyl-CoA isomerases, acyl-CoA dehydrogenase,acyl-CoA oxidase and enoyl-CoA hydratases by subsequent transformationof the transgenic plants produced using the methods and DNA constructsdescribed herein or by traditional plant breeding methods.

[0026] I. Plant Expression Systems

[0027] In a preferred embodiment, the fatty acid oxidation transgenesare expressed from a seed specific promoter, and the proteins areexpressed in the cytoplasm of the developing oilseed. In an alternatepreferred embodiment, fatty acid oxidation transgenes are expressed froma seed specific promoter and the expressed proteins are directed to theplastids using plastid targeting signals. In another preferredembodiment, the fatty acid oxidation transgenes are expressed directlyfrom the plastid chromosome where they have been integrated byhomologous recombination. The fatty acid oxidation transgenes may alsobe expressed throughout the entire plant tissue from a constitutivepromoter. Combinations of tissue specific and constitutive promoterswith the individual genes encoding the enzymes can also be varied toalter the amount and/or location of polymer production. It is alsouseful to be able to control the expression of these transgenes by usingpromoters that can be activated following the application of anagrochemical or other active ingredient to the crop in the field.Additional control of the expression of these genes encompassed by themethods described herein include the use of recombinase technologies fortargeted insertion of the transgenes into specific chromosomal sites inthe plant chromosome or to regulate the expression of the transgenes.

[0028] The methods described herein involve a plant seed having a genomeincluding (a) a promoter operably linked to a first DNA sequence and a3′-untranslated region, wherein the first DNA sequence encodes a fattyacid oxidation polypeptide and optionally (b) a promoter operably linkedto a second DNA sequence and a 3′-untranslated region, wherein thesecond DNA sequence encodes a fatty acid oxidation polypeptide.Expression of the two transgenes provides the plant with a functionalfatty acid β-oxidation system having at least β-ketothiolase,dehydrogenase and hydratase activities in the cytoplasm or plastidsother than peroxisomes or glyoxisomes. The first and/or second DNAsequence may be isolated from bacteria, yeast, fungi, algae, plants, oranimals. It is preferable that at least one of the DNA sequences encodesa polypeptide with at least two, and preferably three, enzymeactivities.

[0029] Transformation Vectors

[0030] DNA constructs useful in the methods described herein includetransformation vectors capable of introducing transgenes into plants.Several plant transformation vector options are available, includingthose described in “Gene Transfer to Plants” (Potrykus, et al., eds.)Springer-Verlag Berlin Heidelberg New York (1995); “Transgenic Plants: AProduction System for Industrial and Pharmaceutical Proteins” (Owen, etal., eds.) John Wiley & Sons Ltd. England (1996); and “Methods in PlantMolecular Biology: A Laboratory Course Manual” (Maliga, et al. eds.)Cold Spring Laboratory Press, New York (1995), which are incorporatedherein by reference. Plant transformation vectors generally include oneor more coding sequences of interest under the transcriptional controlof 5′ and 3′ regulatory sequences, including a promoter, a transcriptiontermination and/or polyadenylation signal, and a selectable orscreenable marker gene. The usual requirements for 5′ regulatorysequences include a promoter, a transcription termination and/or apolyadenylation signal. For the expression of two or more polypeptidesfrom a single transcript, additional RNA processing signals and ribozymesequences can be engineered into the construct (U.S. Pat. No.5,519,164). This approach has the advantage of locating multipletransgenes in a single locus, which is advantageous in subsequent plantbreeding efforts. An additional approach is to use a vector tospecifically transform the plant plastid chromosome by homologousrecombination (U.S. Pat. No. 5,545,818), in which case it is possible totake advantage of the prokaryotic nature of the plastid genome andinsert a number of transgenes as an operon.

[0031] Promoters

[0032] A large number of plant promoters are known and result in eitherconstitutive, or environmentally or developmentally regulated expressionof the gene of interest. Plant promoters can be selected to control theexpression of the transgene in different plant tissues or organelles forall of which methods are known to those skilled in the art (Gasser &Fraley, Science 244:1293-99 (1989)). The 5′ end of the transgene may beengineered to include sequences encoding plastid or other subcellularorganelle targeting peptides linked in-frame with the transgene.Suitable constitutive plant promoters include the cauliflower mosaicvirus 35S promoter (CaMV) and enhanced CaMV promoters (Odell et. al.,Nature, 313: 810 (1985)), actin promoter (McElroy et al., Plant Cell2:163-71 (1990)), AdhI promoter (Fromm et. al., Bio/Technology 8:833-39(1990); Kyozuka et al., Mol. Gen. Genet. 228:40-48 (1991)), ubiquitinpromoters, the Figwort mosaic virus promoter, mannopine synthasepromoter, nopaline synthase promoter and octopine synthase promoter.Useful regulatable promoter systems include spinach nitrate-induciblepromoter, heat shock promoters, small subunit of ribulose biphosphatecarboxylase promoters and chemically inducible promoters (U.S. Pat. No.5,364,780 to Hershey et al.).

[0033] In a preferred embodiment of the methods described herein, thetransgenes are expressed only in the developing seeds. Promoterssuitable for this purpose include the napin gene promoter (U.S. Pat.Nos. 5,420,034 and 5,608,152), the acetyl-CoA carboxylase promoter (U.S.Pat. Nos. 5,420,034 and 5,608,152), 2S albumin promoter, seed storageprotein promoter, phaseolin promoter (Slightom et. al., Proc. Natl.Acad. Sci. USA 80:1897-1901 (1983)), oleosin promoter (Plant et. al.,Plant Mol. Biol. 25:193-205 (1994); Rowley et al., Biochim. Biophys.Acta. 1345:1-4 (1997); U.S. Pat. No. 5,650,554; and PCT WO 93/20216),zein promoter, glutelin promoter, starch synthase promoter, and starchbranching enzyme promoter.

[0034] The transformation of suitable agronomic plant hosts using thesevectors can be accomplished with a variety of methods and plant tissues.Representative plants useful in the methods disclosed herein include theBrassica family including napus, rappa, sp. carinata andjuncea; maize;soybean; cottonseed; sunflower; palm; coconut; safflower; peanut;mustards including Sinapis alba; and flax. Crops harvested as biomass,such as silage corn, alfalfa, or tobacco, also are useful with themethods disclosed herein. Representative tissues for transformationusing these vectors include protoplasts, cells, callus tissue, leafdiscs, pollen, and meristems. Representative transformation proceduresinclude Agrobacterium-mediated transformation, biolistics,microinjection, electroporation, polyethylene glycol-mediated protoplasttransformation, liposome-mediated transformation, and siliconfiber-mediated transformation (U.S. Pat. No. 5,464,765; “Gene Transferto Plants” (Potrykus, et al., eds.) Springer-Verlag Berlin HeidelbergNew York (1995); “Transgenic Plants: A Production System for Industrialand Pharmaceutical Proteins” (Owen, et al., eds.) John Wiley & Sons Ltd.England (1996); and “Methods in Plant Molecular Biology: A LaboratoryCourse Manual” (Maliga, et al. eds.) Cold Spring Laboratory Press, NewYork (1995)).

[0035] II. Methods for Making and Screening for Transgenic Plants

[0036] In order to generate transgenic plants using the constructsdescribed herein, the following procedures can be used to obtain atransformed plant expressing the transgenes subsequent totransformation: select the plant cells that have been transformed on aselective medium; regenerate the plant cells that have been transformedto produce differentiated plants; select transformed plants expressingthe transgene at such that the level of desired polypeptide is obtainedin the desired tissue and cellular location.

[0037] For the specific crops useful for practicing the describedmethods, transformation procedures have been established, as describedfor example, in “Gene Transfer to Plants” (Potrykus, et al., eds.)Springer-Verlag Berlin Heidelberg New York (1995); “Transgenic Plants: AProduction System for Industrial and Pharmaceutical Proteins” (Owen, etal., eds.) John Wiley & Sons Ltd. England (1996); and “Methods in PlantMolecular Biology: A Laboratory Course Manual” (Maliga, et al. eds.)Cold Spring Laboratory Press, New York (1995).

[0038]Brassica napus can be transformed as described, for example, inU.S. Pat. Nos. 5,188,958 and 5,463,174. Other Brassica such as rappa,carinata and juncea as well as Sinapis alba can be transformed asdescribed by Moloney et. al., Plant Cell Reports 8:238-42 (1989).Soybean can be transformed by a number of reported procedures (U.S. Pat.Nos. 5,015,580; 5,015,944; 5,024,944; 5,322,783; 5,416,011; and5,169,770). Several transformation procedures have been reported for theproduction of transgenic maize plants including pollen transformation(U.S. Pat. No. 5,629,183), silicon fiber-mediated transformation (U.S.Pat. No. 5,464,765), electroporation of protoplasts (U.S. Pat. Nos.5,231,019; 5,472,869; and 5,384,253) gene gun (U.S. Pat. Nos. 5,538,877and 5,538,880 and Agrobacterium-mediated transformation (EP 0 604 662Al; PCT WO 94/00977). The Agrobacterium-mediated procedure isparticularly preferred, since single integration events of the transgeneconstructs are more readily obtained using this procedure, which greatlyfacilitates subsequent plant breeding. Cotton can be transformed byparticle bombardment (U.S. Pat. Nos. 5,004,863 and 5,159,135). Sunflowercan be transformed using a combination of particle bombardment andAgrobacterium infection (EP 0 486 233 A2; U.S. Pat. No. 5,030,572). Flaxcan be transformed by either particle bombardment orAgrobacterium-mediated transformation. Recombinase technologies includethe cre-lox, FLP/FRT, and Gin systems. Methods for utilizing thesetechnologies are described for example in U.S. Pat. No. 5,527,695 toHodges et al.; Dale & Ow, Proc. Natl. Acad. Sci. USA 88:10558-62 (1991);Medberry et. al., Nucleic Acids Res. 23:485-90 (1995).

[0039] Selectable Marker Genes

[0040] Selectable marker genes useful in practicing the methodsdescribed herein include the neomycin phosphotransferase gene nptll(U.S. Pat. Nos. 5,034,322 and 5,530,196), hygromycin resistance gene(U.S. Pat. No. 5,668,298), bar gene encoding resistance tophosphinothricin (U.S. Pat. No. 5,276,268). EP 0 530 129 Al describes apositive selection system which enables the transformed plants tooutgrow the non-transformed lines by expressing a transgene encoding anenzyme that activates an inactive compound added to the growth media.Screenable marker genes useful in the methods herein include theβ-glucuronidase gene (Jefferson et. al., EMBO J. 6:3901-07 (1987); U.S.Pat. No. 5,268,463) and native or modified green fluorescent proteingene (Cubitt et. al., Trends Biochem Sci. 20:448-55 (1995); Pang et.al., Plant Physiol. 112:893-900 (1996)). Some of these markers have theadded advantage of introducing a trait, such as herbicide resistance,into the plant of interest, thereby providing an additional agronomicvalue on the input side.

[0041] In a preferred embodiment of the methods described herein, morethan one gene product is expressed in the plant. This expression can beachieved via a number of different methods, including (1) introducingthe encoding DNAs in a single transformation event where all necessaryDNAs are on a single vector; (2) introducing the encoding DNAs in aco-transfonnation event where all necessary DNAs are on separate vectorsbut introduced into plant cells simultaneously; (3) introducing theencoding DNAs by independent transformation events successively into theplant cells i.e. transformation of transgenic plant cells expressing oneor more of the encoding DNAs with additional DNA constructs; and (4)transformation of each of the required DNA constructs by separatetransformation events, obtaining transgenic plants expressing theindividual proteins and using traditional plant breeding methods toincorporate the entire pathway into a single plant.

[0042] III. β-Oxidation Enzyme Pathways

[0043] Production of PHAs in the cytosol of plants requires thecytosolic localization of enzymes that are able to produceR-3-hydroxyacyl CoA thioesters as substrates for PHA synthases. Botheukaryotes and prokaryotes possess a β-oxidation pathway for fatty aciddegradation that consists of a series of enzymes that convert fatty acylCoA thioesters to acetyl CoA. While these pathways proceed viaintermediate 3-hydroxyacyl CoA, the stereochemistry of this intermediatevaries among organisms. For example, the β-oxidation pathways ofbacteria and the peroxisomal pathway of higher eukaryotes degrade fattyacids to acetyl CoA via S-3-hydroxyacyl CoA (Schultz, “Oxidation ofFatty Acids” in Biochemistry ofLipids, Lipoproteins and Membranes (Vanceet al., eds.) pp. 101-06 (Elsevier, Amsterdam 1991)). In Escherichiacoli, an epimerase activity encoded by the β-oxidation multifunctionalenzyme complex is capable of converting S-3-hydroxyacyl CoA toR-3-hydroxyacyl CoA. Yeast possesses a peroxisomal localized fatty aciddegradation pathway that proceeds via intermediate R-3-hydroxyacyl CoA(Hiltunen, et al. J. Biol. Chem. 267: 6646-53 (1992); Filppula, et al. JBiol. Chem. 270:27453-57 (1995)), such that no epimerase activity isrequired to produce PHAs.

[0044] Plants, like other higher eukaryotes, possesses a β-oxidationpathway for fatty acid degradation localized subcellularly in theperoxisomes (Gerhardt, “Catabolism of Fatty Acids [α and β Oxidation]”in Lipid Metabolism in Plants (Moore, Jr., ed.) pp. 527-65 (CRC Press,Boca Raton, Fla. 1993)). Production of PHAs in the cytosol of plantstherefore necessitates the cytosolic expression of a β-oxidationpathway, for conversion of fatty acids to R-3-hydroxyacyl CoA thioestersof the correct chain length, as well as cytosolic expression of anappropriate PHA synthase, to polymerize R-3-hydroxyacyl CoA to polymer.

[0045] Fatty acids are synthesized as saturated acyl-ACP thioesters inthe plastids of plants (Hartwood, “Plant Lipid Metabolism” in PlantBiochemistry (Dey et al., eds.) pp. 237-72 (Academic Press, San Diego1997)). Prior to export from the plastid into the cytosol, the majorityof fatty acids are desaturated via a Δ9 desaturase. The pool of newlysynthesized fatty acids in most oilseed crops consists predominantly ofoleic acid (cis 9-octadecenoic acid), stearic acid (octadecanoic acid),and palmitic acid (hexadecanoic acid). However, some plants, such ascoconut and palm kernel, synthesize shorter chain fatty acids (C8-14).The fatty acid is released from ACP via a thioesterase and subsequentlyconverted to an acyl-CoA thioester via an acyl CoA synthetase located inthe plastid membrane (Andrews, et al., “Fatty acid and lipidbiosynthesis and degradation” in Plant Physiology, Biochemistry, andMolecular Biology (Dennis et al., eds.) pp. 345-46 (Longman Scientific &Technical, Essex, England 1990); Harwood, “Plant Lipid Metabolism” inPlant Biochemistry (Dey et al., eds) p. 246 (Academic Press, San Diego1997)).

[0046] The cytosolic conversion of the pool of newly synthesized acylCoA thioesters via fatty acid degradation pathways and the conversion ofintermediates from these series of reactions to R-3-hydroxyacyl-CoAsubstrates for PHA synthases can be achieved via the enzyme reactionsoutlined in FIG. 1. The PHA synthase substrates are C4-C16R-3-hydroxyacyl CoAs. For saturated fatty acyl CoAs, conversion toR-3-hydroxyacyl CoA thioesters using fatty acids degradation pathwaysnecessitates the following sequence of reactions: conversion of the acylCoA thioester to trans-2-enoyl-CoA (reaction 1), hydration oftrans-2-enoyl-CoA to R-3-hyddroxy acyl CoA (reaction 2a, e.g. yeastsystem operates through this route and the Aeromonas caviae D-specifichydratase yields C4-C7 R-3-hydroxyacyl-CoAs), hydration oftrans-2-enoyl-CoA to S-3-hydroxy acyl CoA (reaction 2b), andepimerization of S-3-hydroxyacyl CoA to R-3-hydroxyacyl CoA (reaction 5,e.g. cucumber tetrafunctional protein, bacterial systems). If3-hydroxyacyl CoA is not polymerized by PHA synthase forming PHA, it canproceed through the remainder of the β-oxidation pathway as follows:oxidation of 3-hydroxyacyl CoA to form β-keto acyl CoA (reaction 3)followed by thiolysis in the presence of CoA to yield acetyl CoA and asaturated acyl CoA thioester shorter by two carbon units (reaction 4).The acyl CoA thioester produced in reaction 4 is free to re-enter theβ-oxidation pathway at reaction 1 and the acetyl-CoA produced can beconverted to R-3-hydroxyacyl CoA by the action of β-ketothiolase(reaction 7) and NADH or NADPH acetoacetyl-CoA reductase (reaction 6).This latter route is useful for producing R-3-hydroxybutyryl-CoA,R-3-hydroxyvaleryl-CoA and R-3-hydroxyhexanoyl-CoA. The R-3-hydroxyacidsof four to sixteen carbon atoms produced by this series of enzymaticreactions can be polymerized by PHA synthases expressed from atransgene, or transgenes in the case of the two subunit synthaseenzymes, into PHA polymers.

[0047] For Δ9 unsaturated fatty acyl CoAs, a variation of the reactionsequences described is required. Three cycles of β-oxidation, asdetailed in FIG. 1, will remove six carbon units yielding an unsaturatedacyl CoA thioester with a cis double bond at position 3. Conversion ofthe cis double bond at position 3 to a trans double bond at position 2,catalyzed by Δ³-cis-Δ²-trans-enoyl CoA isomerase will allow theβ-oxidation reaction sequences outlined in FIG. 1 to proceed. Thisenzyme activity is present on the microbial β-oxidation complexes andthe plant tetrafunctional protein, but not on the yeastfoxl.

[0048] Acyl CoA thioesters also can be degraded to a β-keto acyl CoA andconverted to R-3-hydroxyacyl CoA via a NADH or NADPH dependent reductase(reaction 6).

[0049] Multifunctional enzymes that encode S-specific hydratase,S-specific dehydrogenase, β-ketothiolase, epimerase andΔ³-cis-Δ²-trans-enoyl CoA isomerase activities have been found inbacteria such as Escherichia coli (Spratt, et al., J Bacteriol.158:535-42 (1984)) and Pseudomonas fragi (Immure, et al., J. Biochem.107:184-89 (1990)). The multifunctional enzyme complexes consist of twocopies of each of two subunits such that catalytically active proteinforms a heterotetramer. The hydratase, dehydrogenase, epimerase, andΔ³-cis-Δ²-trans-enoyl CoA isomerase activities are located on onesubunit, whereas the thiolase is located on another subunit. The genesencoding the enzymes from organisms such as E. coli (Spratt, et al., J.Bacteriol. 158:535-42 (1984); DiRusso, J. Bacteriol. 172:6459-68 (1990))and P. fragi (Sato, et al., J. Biochem. 111:8-15 (1992)) have beenisolated and sequenced and are suitable for practicing the methodsdescribed herein. Furthermore, the E. coli enzyme system has beensubjected to site-directed mutagenesis analysis to identify amino acidresidues critical to the individual enzyme activities (He & Yang,Biochemistry 35:9625-30 (1996); Yang et. al., Biochemistry 34:6641-47(1995); He & Yang, Biochemistry 36:11044-49 (1997); He et. al.,Biochemistry 36:261-68 (1997); Yang & Elzinga, J. Biol. Chem.268:6588-92 (1993)). These mutant genes also could be used in someembodiments of the methods described herein.

[0050] Mammals, such as rat, possess a trifunctional β-oxidation enzymein their peroxisomes that contains hydratase, dehydrogenase, andΔ³-cis-Δ²-trans-enoyl CoA isomerase activities. The trifunctional enzymefrom rat liver has been isolated and has been found to be monomeric witha molecular weight of 78 kDa (Palosaari, et al., J Biol. Chem.265:2446-49 (1990)). Unlike the bacterial system, thiolase activity isnot part of the multienzyme protein (Schultz, “Oxidation of Fatty Acids”in Biochemistry of Lipids, Lipoproteins and Membranes (Vance et al.,eds) p. 95 (Elsevier, Amsterdam (1991)). Epimerization in rat occurs bythe combined activities of two distinct hydratases, one which convertsR-3-hydroxyacyl CoA to trans-2-enoyl CoA, and another which convertstrans-2-enoyl CoA to S-3-hydroxyacyl CoA (Smeland, et al., Biochemicaland Biophysical Research Communications 160:988-92 (1989)). Mammals alsopossess β-oxidation pathways in their mitochondria that degrade fattyacids to acetyl CoA via intermediate S-3-hydroxyacyl CoA (Schultz,“Oxidation of Fatty Acids” in Biochemistry of Lipids, Lipoproteins andMembranes (Vance et al., eds) p. 96 (Elsevier, Amsterdam (1991)). Genesencoding mitochondrial β-oxidation activities have been isolated fromseveral animals including a Rat mitochondrial long chain acyl CoAhydratase/3-hydroxy acyl CoA dehydrogenase (GENBANK Accession # D16478)and a Rat mitochondrial thiolase (GENBANK Accession #s DI 3921, D00511).

[0051] Yeast possesses a multifunctional enzyme, Fox2, that differs fromthe β-oxidation complexes of bacteria and higher eukaryotes in that itproceeds via a R-3-hydroxyacyl CoA intermediate instead ofS-3-hydroxyacyl CoA (Hiltunen, et al., J Biol. Chem. 267:6646-53(1992)). Fox2 possesses R-specific hydratase and R-specificdehydrogenase enzyme activities. This enzyme does not possess theΔ³-cis-Δ²-trans-enoyl CoA isomerase activity needed for degradation ofΔ9-cis-hydroxyacyl CoAs to form R-3-hydroxyacyl CoAs. The gene encodingfox2 from yeast has been isolated and sequenced and encodes a 900 aminoacid protein. The DNA sequence of the structural gene and amino acidsequence of the encoded polypeptide is shown in SEQ ID NO:1 and SEQ IDNO:2.

[0052] Plants have a tetrafunctional protein similar to the yeast Fox2,but also encoding a Δ³-cis-Δ²-trans-enoyl CoA isomerase activity (Mulleret., al., J. Biol. Chem. 269:20475-81 (1994)). The DNA sequence of thecDNA and amino acid sequence of the encoded polypeptide is shown in SEQID NO:3 and SEQ ID NO:4.

[0053] IV. Targeting of Enzymes to the Cytoplasm of Oil Seed Crops

[0054] Engineering PHA production in the cytoplasm of plants requiresdirecting the expression of β-oxidation to the cytosol of the plant. Notargeting signals are present in the bacterial systems, such as faoAB.In fungi, yeast, plants, and mammals, β-oxidation occurs in subcellularorganelles. Typically, the genes are expressed from the nuclearchromosome, and the polypeptides synthesized in the cytoplasm aredirected to these organelles by the presence of specific amino acidsequences. To practice the methods described herein using genes isolatedfrom eukaryotic sources, e.g., fatty acid oxidation enzymes fromeukaryotic sources, such as yeast, fungi, plants, and mammals, theremoval or modification of subcellular targeting signals is required todirect the enzymes to the cytosol. It may be useful to add signals fordirecting proteins to the endoplasmic reticulum. Peptides useful in thisprocess are well known in the art. The general approach is to modify thetransgene by inserting a DNA sequence specifying an ER targeting peptidesequence to form a chimeric gene.

[0055] Eukaryotic acyl CoA dehydrogenases, as well as othermitrochondrial proteins, are targeted to the mitochondria via leaderpeptides on the N-terminus of the protein that are usually 20-60 aminoacids long (Horwich, Current Opinion in Cell Biology, 2:625-33 (1990)).Despite the lack of an obvious consensus sequence for mitochondrialimport leader peptides, mutagenesis of key residues in the leadersequence have been demonstrated to prevent the import of themitochrondrial protein. For example, the import of Saccharomycescerevisiae F1 -ATPase was prevented by mutagenesis of its leadersequence, resulting in the accumulation of the modified precursorprotein in the cytoplasm (Bedwell, et al., Mol. Cell Biol. 9:1014-25(1989))

[0056] Three eukaryotic peroxisomal targeting signals have been reported(Gould, et al., J Cell Biol. 108:1657-64 (1989); Brickner, et al., J.Plant Physiol. 113:1213-21 (1997)). The tripeptide targeting signalS/A/C-K/H/1R-L occurs at the C-terminal end of many peroxisomal proteins(Gould, et al., J. Cell Biol. 108:1657-64 (1989)). Mutagenesis of thissequence has been shown to prevent import of proteins into peroxisomes.Some peroxisomal proteins do not contain the tripeptide at theC-terminal end of the protein. For these proteins, it has been suggestedthat targeting occurs via the tripeptide in an internal position withinthe protein sequence (Gould, et al., J Cell Biol. 108:1657-64 (1989)) orvia an unknown, unrelated sequence (Brickner, et al., J. Plant Physiol.113:1213-21 (1997)). The results of in vitro peroxisomal targetingexperiments with fragments of acyl CoA oxidase from Candida tropicalisappear to support the latter theory and suggest that there are twoseparate targeting signals within the internal amino acid sequence ofthe polypeptide (Small, et al., The EMBO Journal 7:1167-73 (1988)). Inthe aforementioned study, the targeting signals were localized to tworegions of 118 amino acids in length, and neither of regions was foundto contain the targeting signal S/A/C-K/H/R-L. A small number ofperoxisomal proteins appear to contain an amino terminal leader sequencefor import into peroxisomes (Brickner, et al., J Plant Physiol.113:1213-21 (1997)). These targeting signals can be deleted or alteredby site directed mutagenesis.

[0057] V. Cultivation and Harvesting of Transgenic Plant

[0058] The transgenic plants can be grown using standard cultivationtechniques. The plant or plant part also can be harvested using standardequipment and methods. The PHAs can be recovered from the plant or plantpart using known techniques such as solvent extraction in conjunctionwith traditional seed processing technologies, as described in PCT WO97/15681, or can be used directly, for example, as animal feed, where itis unnecessary to extract the PHA from the plant biomass.

[0059] Several lines which did not produce seed, produced much higherlevels of biomass. This phenotype therefore may be useful as a means toincrease the amount of green biomass produced per acre for silage,forage, or other biomass crops. End uses include the more cost effectiveproduction of forage crops for animal feed or as energy crops forelectric power generation. Other uses include increasing biomass levelsin crops, such as alfalfa or tobacco, for subsequent recovery ofindustrial products, such as PHAs by extraction.

[0060] The compositions and methods of preparation and use thereofdescribed herein are further described by the following non-limitingexamples.

EXAMPLE 1

[0061] Isolation and Characterization of the Pseudomonas putida faoABGenes and Fao Enzyme

[0062] All DNA manipulations, including PCR, DNA sequencing E. colitransformation, and plasmids purification, were performed using standardprocedures, as described, for example, by Sambrook et. al., MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, NewYork (1989)). The genes encoding faoAB from Pseudomonas putida wereisolated using a probe generated from P. putida genomic DNA by PCR(polymerase chain reaction) using primers 1 and 2 possessing homology tofaoB from Pseudomonas fragi (Sato, et al., J. Biochem. 111:8-15 (1992)).

[0063] Primer 1: Primer 1: 5′ gat ggg ccg ctc caa ggg tgg 3′ (SEQ IDNO:5) Primer 2: 5′ caa ccc gaa ggt gcc gcc att 3′ (SEQ ID NO:6)

[0064] A 1.1 kb DNA fragment was purified from the PCR reaction and usedas a probe to screen a P. putida genomic library constructed in plasmidpBKCMV using the lambda ZAP expression system (Stratagene). PlasmidpMFX1 was selected from the positive clones and the DNA sequence of theinsert containing thefaoAB genes and flanking sequences determined. Thisis shown in SEQ ID NO:7. A fragment containingfaoAB was subcloned withthe native P. putida ribosome binding site intact into the expressionvector pTRCN forming plasmid pMFX3 as follows. Plasmid pMFX1 wasdigested with BsrG I. The resulting protruding ends were filled in withKlenow. Digestion with Hind III yielded a 3.39 kb blunt ended/Hind IIIfragment encoding FaoAB. The expression vector pTRCN was digested withSma I/Hind III and ligated with thefaoAB fragment forming the 7.57 kbplasmid pMFX3.

[0065] Enzymes in the FaoAB multienzyme complex were assayed as follows.Hydratase activity was assayed by monitoring the conversion of NAD toNADH using the coupling enzyme L-p-hydroxyacyl CoA dehydrogenase aspreviously described, except that assays were run in the presence of CoA(Filppula, et al., J. Biol. Chem. 270:27453-57 (1995)). Severe productinhibitation of the coupling enzyme was observed in the absence of CoA.The assay contained (1 mL final volume) 60 μM crotonyl CoA, 50 μMTris-CI, pH 9, 50 μg bovine serum albumin per mL, 50 mM KCl, 1 mM NAD, 7μg L-specific β-hydroxyacyl CoA dehydrogenase from porcine heart per mL,and 0.25 mM CoA. The assay was initiated with the addition of FaoAB tothe assay mixture. A control assay was perfonned without substrate todetermine the rate of consumption of NAD in the absence of -thehydratase generated product, S-hydroxybutyryl CoA. One unit of activityis defined as the consumption of one μMol of NAD per min (ε₃₄₀=6220M⁻¹cm⁻¹).

[0066] Hydroxyacyl CoA dehydrogenase was assayed in the reversedirection with acetoacetyl CoA as the substrate by monitoring theconversion of NADH to NAD at 340 nm (Binstock, et al., Methods inEnzymology, 71:403 (1981)). The assay contained (1 mL final volume) 0.1M KH₂PO₄, pH 7, 0.2 mg bovine serum albumin per mL, 0.1 mM NADH, and 33μM acetoacetyl CoA. The assay was initiated with the addition of FaoABto the assay mixture. When necessary, enzyme samples were diluted in 0.1M KH₂PO₄, pH 7, containing 1 mg bovine serum albumin per mL. A controlassay was performed without substrate acetoacetyl CoA to detect the rateof consumption of NADH in the crude due to enzymes other thanhydroxyacyl CoA dehydrogenase. One unit of activity is defined as theconsumption of one μMol of NADH per minute (ε₃₄₀=6220 M⁻¹cm⁻¹).

[0067] HydroxyacylCoA dehydrogenase was assayed in the forward directionwith crotonyl CoA as a substrate by monitoring the conversion of NAD toNADH at 340 nm (Binstock, et al., Methods in Enzymology, 71:403 (1981)).The assay mixture contained (1 mL final volume) 0.1 M KH₂PO₄, pH 8, 0.3mg bovine serum albumin per mL, 2 mM β-mercaptoethanol, 0.25 mM CoA, 30μM crotonyl CoA, and an aliquot of FaoAB. The reaction was preincubatedfor a couple of minutes to allow in situ formation of S-hydroxybutyrylCoA. The assay then was initiated by the addition of NAD (0.45 mM). Acontrol assay was performed without substrate to detect the rate ofconsumption of NAD due to enzymes other than hydroxyacyl CoAdehydrogenase. One unit of activity is defined as the consumption of oneμMol of NAD per minute (ε₃₄₀=6220 M⁻¹cm⁻¹).

[0068] Thiolase activity was determined by monitoring the decrease inabsorption at 304 nm due to consumption of substrate acetoacetyl CoA aspreviously described with some modifications (Palmer, et al., J. Biol.Chem. 266:1-7 (1991)). The assay contained (final volume 1 mL) 62.4 mMTris-Cl, pH 8.1, 4.8 mM MgCl₂, 62.5 μM CoA, and 62.5 μM acetoacetyl CoA.The assay was initiated with the addition of FaoAB to the assay mixture.A control sample without enzyme was performed for each assay to detectthe rate of substrate degradation of pH 8.1 in the absence of enzyme.One unit of activity is defined as the consumption of one μMol ofsubstrate acetoacetyl CoA per minute (ε₃₄₀=16900 M⁻¹cm⁻¹).

[0069] Epimerase activity was assayed as previously described (Binstock,et al., Methods in Enzymology, 71:403 (1981)) except thatR-3-hydroxyacyl CoA thioesters were utilized instead ofD,L-3-hydroxyacyl CoA mixtures. The assay contained (final volume 1 mL)30 μM R-3-hydroxyacyl CoA, 150 mM KH₂PO₄ (pH 8), 0.3 mg/mL BSA, 0.5 mMNAD, 0.1 mM CoA, and 7 μg/mL L-specific β-hydroxyacyl CoA dehydrogenasefrom porcine heart. The assay was initiated with the addition of FaoAB.

[0070] For expression of FaoAB in DH5α/pMFX3, cultures were grown in2×TY medium at 30° C. 2×TY medium contains (per L) 16 g tryptone, 10 gyeast, and 5 g NaCl. A starter culture was grown overnight and used toinoculate (1% inoculum) fresh medium (100 mL in a 250 mL Erlenmeyerflask for small scale growths; 1.5 L in a 2.8 L flask for large scalegrowths). Cells were induced with 0.4 mM IPTG when the absorbance at 600nm was in the range of 0.4 to 0.6. Cells were cultured an additional 4 hprior to harvest. Cells were lysed by sonication, and the insolublematter was removed from the soluble proteins by centrifugation. Acyl CoAdehydrogenase activity was monitored in the reverse direction to ensureactivity of the FaoA subunit (SEQ ID NO:31) and thiolase activity wasassayed to determine activity of the Fao subunit. FaoAB in DH5α/pMFX3contained dehydrogenase and thiolase activity values of 4.3 and 0.99U/mg, respectively, which is significantly more than the 0.0074 and0.0033 U/mg observed for dehydrogenase and thiolase, respectively, incontrol strain DH5α/pTRCN.

[0071] FaoAB was purified from DH5α/pMFX3 using a modified procedurepreviously described for the purification of FaoAB from Pseudoinonasfragi (Imamura, et al., J. Biochem. 107:184-89 (1990)). Thiolaseactivity (assayed in the forward direction) and dehydrogenase activities(assayed in the reverse direction) were monitored throughout thepurification. Three liters of DH5α/pMFX3 cells (2×1.5 L aliquots in 2.8L Erlenmeyer flasks) were grown in 2×TY medium using the cell growthprocedure previously described for preparing cells for enzyme activityanalysis. Cells (15.8 g) were resuspended in 32 mL of 10 mM KH₂PO₄, pH7, and lysed by sonication. Soluble proteins were removed from insolublecells debris by centrifugation (18,000 RPM, 30 min., 4° C.). The solubleextract was made 50% in acetone and the precipitated protein wasisolated by centrifugation and redissolved in 10 mM KH₂PO4, pH 7. Thesample was adjusted to 33% saturation with (NH₄)₂SO₄ and the soluble andinsoluble proteins were separated by centrifugation. The resultingsupernatant was adjusted to 56% saturation with (NH₄)₂SO₄ and theinsoluble pellet was isolated by centrifugation and dissolved in 10 mMKH₂PO₄, pH 7. The sample was heated at 50° C. for 30 min. and thesoluble proteins were isolated by centrifugation and dialyzed in a 6,000to 8,000 molecular weight cut off membrane in 10 mM KH₂PO₄, pH 7 (2×3 L;20 h). The sample was loaded on a Toyo Jozo DEAE FPLC column (3 cm x 14cm) that previously had been equilibrated in 10 mM KH₂PO₄, pH 7. Theprotein was eluted with a linear gradient (100 mL by 100 mL; 0 to 500 mMNaCl in 10 KH₂PO₄, pH 7) at a flow of 3 mL/min. FaoAB eluted between 300and 325 mM NaCl. The sample was dialyzed in a 50,000 molecular weightcut off membrane in 10 mM KH₂PO₄, pH 7 (1×2 L; 15h) prior to loading ona macro-prep hydroxylapatite 18/30 (Biorad) FPLC column (2 cm×15 cm)that previously had been equilibrated in 10 mM KH₂PO₄, pH 7. The proteinwas eluted with a linear gradient (250 mL by 250 mL; 10 to 500 mMKH₂PO₄, pH 7) at a flow rate of 3 mL/min. FaoAB eluted between 70 and130 mM KH₂PO₄. The fractions containing activity were concentrated to 9mL using a MILLIPORE™ 100,000 molecular weight cutoff concentrator. Thebuffer was exchanged 3 times with 10 mM KH₂PO₄, pH 7 containing 20%sucrose and frozen at −70° C. Enzyme activities of the hydroxylapatitepurified fraction were assayed with a range of substrates. The resultsare shown in Table 1 below. TABLE 1 Enzyme Substrates and ActivitiesEnzyme Substrate Activity (U/mg) hydratase crotonyl CoA 8.8dehydrogenase (forward) crotonyl CoA 0.46 dehydrogenase (reverse)acetoacetyl CoA 29 thiolase acetoacetyl CoA 9.9 epimeraseR-3-hydroxyoctanyl CoA 0.022 epimerase R-3-hydroxyhexanyl CoA 0.0029epimerase R-3-hydroxybutyryl CoA 0.000022

EXAMPLE 2

[0072] Production of Antibodies to the FaoAB and FaoAB Polypeptides

[0073] Following purification of the FaoAB protein as described inExample 1, a sample was separated by SDS-PAGE. The protein bandcorresponding to the FaoA (SEQ ID NO:31) and FaoB (SEQ ID NO:26) wasexcised and used to immunize New Zealand white rabbits with completeFreunds adjuvant. Boosts were performed using incomplete Freunds atthree week intervals. Antibodies were recovered from serum by affinitychromatography on Protein A columns (Pharmacia) and tested against theantigen by Western blotting procedures. Control extracts of Brassicaseeds were used to test for cross reactivity to plant proteins. No crossreactivity was detected.

EXAMPLE 3

[0074] Construction of Plasmids for Expression of the Pseudomonas putidofao AB Genes in Transgenic Oilseeds

[0075] Construction of pSBS2024

[0076] Oligonucleotide primers GVR471 GVR471 5′-CGGTACCCATTGTACTCCCAGTATCAT-3′ and (SEQ ID NO:8) GVR472 5′-CATTTAAATAGTAGAGTATTGAATATG-3′ (SEQ ID NO:9)

[0077] homologous to sequences flanking the 5′ and 3′ ends (underlined),respectively, of the bean phaseolin promoter (SEQ ID NO:10; Slightom etal., 1983) were designed with the addition of Kpizl (in italics,nucleotides 1-7 in SEQ ID NO:8) and SwaI (in italics, nucleotides 1-9 inSEQ ID NO:9) at the 5′ ends of GVR471 and GVR472, respectively. Theserestriction sites were incorporated to facilitate cloning. The primerswere used to amplify a 1.4 kb phaseolin promoter, which was cloned atthe SmaI site in pUC19 by blunt ended ligation. The designated plasmid,pCPPI (see FIG. 2) was cut with SalI and SwaI and ligated to a SalI/SwaIphaseolin terminator (SEQ ID NO:27). The bean phaseolin terminatorsequence encompassing the polyadenylation signals was amplified usingthe following PCR primers: GVR396: GVR396: (SEQ ID NO:22)5′-GATTTAAATGCAAGCTTAAATAAGTATGAACTAAAATGC-3′ and GVR397: (SEQ ID NO.23)5′-CGGTACCTTAGTTGGTAGGGTGCTA-3′

[0078] and the 1.2Kb fragment (SEQ ID NO:27) cloned into Sall-Sal siteof pCCP1 to obtain pSBS2024 (FIG. 2). The resulting plasmid whichcontains a unique HindIII site for cloning was called pSBS2024 (FIG. 2).

[0079] Construction of pSBS2025

[0080] A soybean oleosin promoter fragment (SEQ ID NO:11; Rowley et al.,1997) was simplified with primers that flank the DNA sequence.

[0081] Primer JA408 (SEQ ID NO:12) 5′-TCTAGATACATCCATTTCTTAATATAATCCTCTTATTC-3′

[0082] contains sequences that are complementary to the 5′ end(underlined).

[0083] Primer np1 5′-CATTTAAT CGTTAAGGTGAAGGTAGGGCT-3′ (SEQ ID NO:13)

[0084] contains sequences homologous to the 3′ end (underlined) of thepromoter fragment. The restriction sites Xbal (in italics) and Swal (initalics) were incorporated at the 5′ end of JA408 and np1, respectively,to facilitate cloning. The primers were used to amplify a 975 bppromoter fragment, which then was cloned into Sniall site ofpUC19 (seeFIG. 2). The resulting plasmid, pCSPI, was cut with SalI and SwaI andligated to the soybean terminator (SEQ ID NO:28). The soybean oleosinterminator was amplified by PCR using the following primers:

[0085] JA410: (SEQ ID NO:29) 5′-AAGCTTACGTGATGAGTATTAATGTGTTGTTATG-3′

[0086] and

[0087] JA411: (SEQ ID NO:30) 5′-TCTAGACAATTCATCAAATACAAATCACATTGCC-3′

[0088] and the 225 bp fragment cloned into the SalI-SwaI site of pCSP1to obtain plasmid pSBS2025 (FIG. 6). The designated plasmid, pSBS2025,carried a unique HindIII site for cloning (FIG. 2).

[0089] Construction of Promoter-coding Sequence Fusions

[0090] Two oligonucleotide primers were synthesized:

[0091] np2 5′AAGCTT AAA ATGATTTACGAAGGTAAAGCC-3′ (SEQ ID NO:14)

[0092] homologous to nucleotides 553 to 573 of the 5′ flankingsequences, and

[0093] np3 5′ATTGCTTTCAGTTGAAGCGCTG-3′ (SEQ ID NO:15)

[0094] complementary to nucleotides 2700 to 2683 flanking the 3′ end ofmf1 (faoA, SEQ ID NO:24) of plasmid pmfx3. A HindIII (in italics) sitewas introduced at the 5′ end of primers np2 and np3 to facilitatecloning. In addition, a 3 bp AAA sequence (bold) was incorporated toobtain a more favorable sequence surrounding the plant translationalinitiation codon. Primers np2 and np3 were used to amplify the fragmentand cloned into SmaI site of pUC19. The resulting plasmid was calledpCmf1 (FIGS. 3A and 3B). Plasmid pBmf2 was constructed in a similarprocess (FIGS. 5A and 5B). In order to generate a HindIII (in italics)at 5′ and 3′ ends of the mf2 (faoB) gene (SEQ ID NO:25) for cloning, asecond set of synthetic primers were designed.

[0095] Primers np4 5′-AAGCTTAAA ATGAGCCTGAATCCAAGAGAC-3′ (SEQ ID NO:16)

[0096] complementary to 5′ (nucleotides 2732-2752 bp) and np5 5′-AAGCTTTCAGACGCGTTCGAAGACAGTG-3′ (SEQ ID NO:17)

[0097] homologous to 3′ (nucleotides 3907-3886 bp) sequences of mf2(faoB, SEQ ID NO:25) of plasmid pmfx3 were used in a PCR reaction toamplify the 1.17 kb DNA fragment. The resulting PCR product was clonedinto the EcoR V site of pBluescript. The plasmid was referred to aspBmf2.

[0098] Both plasmids were individually cut with HindIII and theirinserts cloned in plasmids pSBS2024 and pSBS2025, which had previouslybeen linearized with the same restriction enzyme. As a result, thefollowing plasmids were generated: pmf124 and pmf125 (FIGS. 3A and 3B)and pmf224 and pmf225 (FIGS. 5A and 5B) containing the Fao genes (mf1and mf2) fused to either the phaseolin or soybean promoters. DNAsequence analysis confirmed the correct promoter-codingsequence-termination sequence fusions for pmf124, pmf125, pmf224, andpmf225.

EXAMPLE 4

[0099] Assembly of Promoter-coding Sequence Fusions into PlantTransformation Vectors

[0100] After obtaining plasmids pmf124, pmf125, pmf224, and pmf225,promoter-coding sequence fusions were independently cloned into thebinary vectors, pCGN1559 (McBride and Summerfelt, 1990) containing theCaMV 35S promoter driving the expression of NPTII gene (conferringresistance to the antibiotic kanamycin) and pSBS2004 containing aparsley ubiquitin promoter driving the PPT gene, which confersresistance to the herbicide phosphinothricine. Binary vectors suitablefor this purpose with a variety of selectable markers can be obtainedfrom several sources.

[0101] The phaseolin-mf21 fusion cassette was released from the parentplasmid with XbaI and ligated with pCGN1559, which had been linearizedwith the same restriction enzyme. The resulting plasmid was designatedpCGmf124 (FIGS. 3A and 3B). Plasmid pCGmf125 containing the soybean-mf1fusion was constructed in a similar way (FIGS. 3A and 3B), except thatboth pmf125 and pCGN1559 were cut with BamHI before ligation.

[0102] Construction of pmf1249 an pmf1254

[0103] The plasmid pSBS²004 was linearized with BamHI fragmentcontaining the soybean-mf1 fusion. This plasmid was designated pmf1254(FIGS. 4A and 4B). Similarly, the XbaI phaseolin-mfl fusion fragment wasligated to pSBS2004 which had been linearized with the same restrictionenzyme. The resulting plasmid was designated pmf1249 (FIGS. 4A and 4B).

[0104] Construction of pCGmf224 and pCGmf225

[0105] The phaseolin-mf2 and soybean-mf2 fusions were constructed byexcising the fusions from the vector by cutting with either BamHI orXbaI, and cloned into pCGN1559 which had been linearized with eitherrestriction enzyme (FIGS. 5A and 5B).

[0106] Construction of pCGmf1P2S and pCGmf2P1S

[0107] The two expression cassettes containing the promoter-codingsequence fusions were assembled on the same binary vector as follows:Plasmid pmf124 containing the phaseolin-mf1 fusion was cut with BamHIand cloned into the BamHI site ofpCGN1559 to create pCGmfB124. Thisplasmid then was linearized with XbaI and ligated to the XbaI fragmentof pmf225 containing the soybean-mf2 fusion. The final plasmid wasdesignated pCGmf1P2S (FIGS. 6A and 6B). Plasmid pCGmf2P1S was assembledin similar manner. The phaseolin-mf2 fusion was released from pmf224 bycutting with BamHI and cloned at the BamHI site ofpCGN1559. Theresulting plasmid, pCGmfB224, was linearized with XbaI and ligated tothe XbaI fragment of pmf125 containing the soybean-mf1 fusion (FIGS. 6Aand 6B).

EXAMPLE 5

[0108] Transformation of Brassica

[0109] Brassica seeds were surface sterilized in 10% commercial bleach(Javex, Colgate-Palmolive) for 30 min. with gentle shaking. The seedswere washed three times in sterile distilled water. Seeds were placed ingermination medium comprising Murashige-Skoog (MS) salts and vitamins,3% (w/v) sucrose and 0.7% (w/v) phytagar, pH 5.8 at a density of 20 perplate and maintained at 24° C. and a 16 h light/8 h dark photoperiod ata light intensity of 60-80 μm⁻²s⁻¹ for four to five days.

[0110] Each of the constructs, pCGmf124, pCGmf125, pCGmf224, pCGmf1P2S,and pCGmf2P1S were introduced into Agrobacterium tumefacians strainEHA101 (Hood et al., J. Bacteriol. 168:1291-1301 (1986)) byelectroporation. Prior to transformation of cotyledonary petioles,single colonies of strain EHA101 harboring each construct were grown in5 ml of minimal medium supplemented with 100 mg kanamycin per liter and100 mg gentamycin per liter for 48 hr at 28° C. One milliliter ofbacterial suspension was pelletized by centrifugation for 1 min in amicrofuge. The pellet was resuspended in 1 ml minimal medium.

[0111] For transformation, cotyledons were excised from 4 day old, or insome cases 5 day old, seedlings, so that they included approximately 2mm of petiole at the base. Individual cotyledons with the cut surface oftheir petioles were immersed in diluted bacterial suspension for 1 s andimmediately embedded to a depth of approximately 2 mm in co-cultivationmedium, MS medium with 3% (w/v) sucrose and 0.7% phytagar and enrichedwith 20 μM benzyladenine. The inoculated cotyledons were plated at adensity of 10 per plate and incubated under the same growth conditionsfor 48 h. After co-cultivation, the cotyledons then were transferred toregeneration medium comprising MS medium supplemented with 3% sucrose,20 μM benzyladenine, 0.7% (w/v) phytagar, pH 5.8, 300 mg timentinin perliter, and 20 mg kanamycin sulfate per liter.

[0112] After two to three weeks, regenerant shoots obtained were cut andmaintained on “shoot elongation” medium (MS medium containing, 3%sucrose, 300 mg timentin per liter, 0.7% (w/v) phytagar, 300 mgtimentinin per liter, and 20 mg kanamycin sulfate per liter, pH 5.8) inMagenta jars. The elongated shoots were transferred to “rooting” mediumcomprising MS medium, 3% sucrose, 2 mg indole butyric acid per liter,0.7% phytagar, and 500 mg carbenicillin per liter. After roots emerged,plantlets were transferred to potting mix (Redi Earth, W.R. Grace andCo.). The plants were maintained in a misting chamber (75% relativehumidity) under the same growth conditions. Two to three weeks aftergrowth, leaf samples were taken for neomycin phosphotransferase (NPTII)assays (Moloney et al., Plant Cell Reports 8:238-42 (1989)).

[0113] Seeds from the FaoA and FaoB transgenic lines can be analyzed forexpression of the fatty acid oxidation polypeptides by western blottingusing the anti-FaoA and anti-FaoB antibodies. The FaoB polypeptide (SEQID NO:26) is not functional in the absence of the FaoA gene product;however, the FaoAB gene product has enzyme activity.

[0114] Transgenic lines expressing the FaoA and FaoB complex areobtained by crossing the FaoA and FaoB transgenic lines expressing theindividual polypeptides and seeds analyzed by western blotting andenzymes assays as described.

EXAMPLE 6

[0115] Transformation of B. napus cv. Westar and Analysis of TransgenicLines

[0116] Transformation

[0117] The protocol used was adopted from a procedure described byMoloney et al. (1989). Seeds of Brassica napus cv. Westar were surfacesterilized in 10% commercial bleach (Javex, Colgate-Palmolive CanadaInc.) for 30 min with gentle shaking. The seeds were washed three timesin sterile distilled water. Seeds were placed on germination mediumcomprising Murashige-Skoog (MS) salts and vitamins, 3% sucrose and 0.7%phytagar, pH 5.8 at a density of 20 per plate and maintained at 24° C.in a 16 h light/8 h dark photoperiod at a light intensity of 60-80μEm⁻²s⁻¹ for four to five days.

[0118] Each of the constructs, pCGmf124, pCGmf125, pCGmf224, pCGmf225,pCGmf1P2S, and pCGmf2P1S were introduced into Agrobacterium tumefaciensstrain EHA101 (Hood et al. 1986) by electroporation. Prior totransformation of cotyledonary petioles, single colonies of strainEHAIOI harboring each construct were grown in 5 mL of minimal mediumsupplemented with 100 mg kanamycin per liter, and 100 mg gentamycin perliter for 48 h at 28° C. One milliliter of bacterial suspension waspelletized by centrifugation for 1 min in a microfuge. The pellet wasresuspended in 1 mL minimal medium.

[0119] For transformation, cotyledons were excised from four-day-old, orin some cases five-day-old, seedlings so that they includedapproximately 2 mm of petiole at the base. Individual cotyledons withthe cut surface of their petioles were immersed in diluted bacterialsuspension for 1 s and immediately embedded to a depth of approximately2 mm in co-cultivation medium, MS medium with 3% sucrose and 0.7%phytagar, enriched with 20 μM benzyladenine. The inoculated cotyledonswere plated at a density of 10 per plate and incubated under the samegrowth conditions for 48 h. After co-cultivation, the cotyledons thenwere transferred to regeneration medium, which comprised MS mediumsupplemented with 3% sucrose, 20 μM benzyladenine, 0.7% phytagar, pH5.8, 300 mg timentinin per liter, and 20 mg kanamycin sulfate per liter.

[0120] After two to three weeks, regenerant shoots were obtained, cut,and maintained on “shoot elongation” medium (MS medium containing 3%sucrose, 300mg timentin per liter, 0.7% phytagar, and 20 mg kanamycinper liter, pH 5.8) in Magenta jars. The elongated shoots then weretransferred to “rooting” medium, which comprised MS medium, 3% sucrose,2 mg indole butyric acid per liter, 0.7% phytagar and 500 mgcarbenicillin per liter. After roots emerged, the plantlets weretransferred to potting mix (Redi Earth, W.R. Grace and Co. Canada Ltd.).The plants were maintained in a misting chamber (75% RH) under the samegrowth conditions. Two to three weeks after growth, leaf samples weretaken for neomycin phosphotransferase (NPT II) assays (Moloney et al.1989). The results are shown in Table 2 below. The data show the numberof plants that were confinned to be transformed. TABLE 2 NPT II Activityin Transformed Plants No. of plants No. of NPTII NPTII confirmedConstructs plants assayed confirmed transformed ¹pCGmf124 47 27 23 33²pCGmf125 37 28 18 18 ³pCGmf224 49 40 30 39 ⁴pCGmf225 52 37 28 34⁵pCGmf1P2S 27 27 21 21 ⁶pCGmf2P1S

[0121] The fate of the transforming DNA was investigated for sixteenrandomly selected transgenic lines. Southern DNA hybridization analysisshowed that the FaoA and/or FaoB were integrated into the genomes of thetransgenic lines tested.

[0122] Approximately 80% of the pmf124 transgenic plants in which theFaoA gene is expressed from the strong bean phaseolin promoter wereobserved to be male sterile. Clearly high level expression of the FaoAgene from this promoter results in functional expression of the FaoAgene product which impairs seed and/or pollen development. This resultwas very unexpected, since it was not anticipated that the plant cellswould be capable of carrying out the first step in the β-oxidationpathway in the cytosol. This result, however, provides additionalapplications for expressing β-oxidation genes in plants for malesterility for hybrid production or to prevent the production of seed. Itwas also note that in a side-by-side comparison with normal transgeniclines, the pmf124 lines produced much higher levels of biomass,presumably due to the elimination of seed development. This phenotypetherefore may be useful as a means to increase the amount of greenbiomass produced per acre for silage, forage, or other biomass crops.Here, the use of an inducible promoter system or recombinase technologycould be used to produce seed for planting. Seven of the sterile plantswere successfully cross-pollinated with pollen from pmf225 transgeniclines and set seeds.

[0123] Northern analysis on RNA from seeds from pmf224 lines containingthe phaseolin promoter-FaoB constructs showed a signal indicative of theexpected 1.2 kb transcript in all the samples tested except the control.Northern analysis on RNA from seeds from pmf125 lines containing theweak soybean oleolsin promoter-FaoA constructs revealed a transcript ofthe expected size of 2.1 kb. Western blotting on 300-500 μg of proteinfrom approximately 80% of seeds of pmf125 plants where the FaoA gene isexpressed from the relatively weak soybean oleosin promoter wereinconclusive, although a weak signal was detected in one transgenicline.

[0124] Fatty Acid Analysis

[0125] Given the unexpected results indicating a strong metabolic effectof expressing the FaoA gene from the strong bean phaseolin promoter inseeds, the fatty acid profile of the seeds from transgenic linesexpressing the FaoA gene from the weak soybean oleosin promoter wasanalyzed. Seeds expressing only the FaoA gene or also expressing theFaoB gene from the bean phaseolin promoter were examined. The analysiswas carried out as described in Millar et al., The Plant Cell11:1889-902 (1998). Seed fatty acid methyl esters (FAMES) were preparedby placing ten seeds of B. napus in 15 x 45-mm screw capped glass tubesand heating at 80° C. in 0.75 mL of IN methanolic HCl reagent (Supelco,PA) and 10 μL of 1 mg 17:0 methyl ester (internal standard) per mLovernight. After cooling the samples, the FAMES were extracted with 0.3mL hexane and 0.5 mL 0.9% NaCl by vortexing vigorously. The samples wereallowed to stand to separate the phases, and 300 μL of the organic phasewas drawn and analyzed on a Hewlett-Packard gas chromatograph.

[0126] Fatty acid profile analysis indicated the presence of anadditional component or enhanced component in the lipid profile in allof the transgenic plants expressing the FaoA gene SEQ ID NO:24 which wasabsent from the control plants. This result again proves conclusivelythat the FaoA gene is being transcribed and translated and that the FaoApolypeptide SEQ ID NO:27 is catalytically active. This peak also wasobserved in eleven additional transgenic plants harboring SoyP-FaoA,PhaP-FaoA-SoyP-FaoB, SoyP-FaoA-PhaP-FaoB genes and a sterile (PhaP-FaoA)plant cross-pollinated with SoyP-FaoB. These data clearly demonstratefunctional expression of the FaoA gene and that even the very low levelsof expression are sufficient to change the lipid profile of the seed.Adapting the methods described herein, one of skill in the art canexpress these genes at levels intermediate between that obtained withthe phaseolin promoter and the soybean oleosin promoter using otherpromoters such as the Arabidopsis oleosin promoter, napin promoter, orcruciferin promoter, and can use inducible promoter systems orrecombinase technologies to control when fatty acid oxidation transgenesare expressed.

EXAMPLE 7

[0127] Yeast β-oxidation Multi-functional Enzyme Complex

[0128]S. cerevisiae contains a β-oxidation pathway that proceeds viaR-hydroxyacyl CoA rather than the S-3-hydroxyacyl CoA observed inbacteria and higher eukaryotes. The fox2 gene from yeast encodes ahydratase that produces R-3-hydroxyacyl CoA from trans-2-enoyl-CoA and adehydrogenase that utilizes R-3-hydroxyacyl-CoA to produce β-keto acylCoAs.

[0129] The fox2 gene (sequence shown in SEQ ID NO:1) was isolated fromS. cerevisiae genomic DNA by PCR in two pieces. Primers N-fox2b andN-bamfox2b were utilized to PCR a 1.1 kb SmaI/BamHI fragment encodingthe N-terminal region of Fox2, and primers C-fox2 and C-bamfox2 wereutilized to PCR a 1.6 kb BamHI/XbaI fragment encoding the C-terminalFox2 region. The full fox2 gene was reconstructed via subcloning invector pTRCN. N-fox2b     fox2       tcc ccc ggg agg agg ttt tta tta tgcctg gaa att tat cct tca aag ata gag tt (SEQ ID NO:18) N-bamfox2b  fox2      aaggatccttgatgtcatttacaactacc (SEQ ID NO:19) C-fox2      fox2      gct cta gat agg gaa aga tgt atg taa g (SEQ ID NO:20)C-bamfox2   fox2       tgacatcaaggatcctttt (SEQ ID NO:21)

[0130] The fox1 gene, however, does not possess a β-ketothiolaseactivity and this activity must be supplied by a second transgene.Representative sources of such a gene include algae, bacteria, yeast,plants, and mammals. The bacterium Alcaligenes eutrophus possesses abroad specificity β-ketothiolase gene suitable for use in the methodsdescribed herein. It can be readily isolated using the acetoacetyl-CoAthiolase gene as a hybridization probe, as described in U.S. Pat. No.5,661,026 to Peoples et al. This enzyme also has been purified (Haywoodet al., FEMS Micro. Lett. 52:91 (1988)), and the purified enzyme isuseful for preparing antibodies or determining protein sequenceinformation as a basis for the isolation of the gene.

EXAMPLE 8

[0131] Plant β-Oxidation Gene

[0132] The DNA sequence of the cDNA encoding β-oxidation tetrafunctionalprotein, shown in SEQ ID NO:4, can be isolated as described inPreisig-Muller et al., J. Biol. Chem. 269:20475-81 (1994). Theequivalent gene can be isolated from other plant species includingArabidopsis, Brassica, soybean, sunflower, and corn using similarprocedures or by screening genomic libraries, many of which arecommercially available, for example from Clontech Laboratories Inc.,Palo Alto, Calif., USA. A peroxisomal targeting sequence P-R-M wasidentified at the carboxy terminus of the protein. Constructs suitablefor expressing in the plant cytosol can be prepared by PCR amplificationof this gene using primers designed to delete this sequence.

EXAMPLE 9

[0133] Expression of PHA Biosynthetic Pathways in Seeds of Brassicanapus.

[0134] Synthesis of PHAs via β-oxidation requires a reductase for thereduction of acetoacetyl-CoA and a PHA synthase for subsequentpolymerization of the resulting hydroxyacyl-CoA molecules. To expressFaoA, FaoB, reductase and synthase in plants, the promoters from beanphaseolin (pha), soybean oleosin (soy) and Arabidopsis oleosin (Ara)were used to express the bacterial genes in a seed-specific manner. Inaddition, a constitutive parsley ubiquitin (ubiq) regulatory sequencewas used to express the synthase gene.

[0135] Seed-Specific-FaoA and FaoB Constructs

[0136] For seed-specific expression of the bacterial FaoA, FaoB,reductase and synthase genes, and constitutive expression of thesynthase gene, plant promoter-terminator cassettes were constructed. Allthe expression cassettes were constructed in pBluescript beforesubcloning in Agrobacterium-based plant transformation vector.

[0137] The Pseudomonas putida FaoA and FaoB genes were amplified fromplasmid pMFX3, cloned into pUC19 and pBluescript respectively, andsequenced. Functional assays using the amplified FaoA (mf1) geneperformed at Metabolix Inc. found the PCR fragment to contain codingsequence which specifies biological activities for hydratase,dehydrogenase and thiolase. The FaoA (mf1) and FaoB (mf2) PCR fragmentswere inserted into an expression cassette containing phaseolin(pSBS2024), soybean oleosin (pSBS2025) or Arabidopsis oleosin (pSBS2038)regulatory sequences shown.

[0138] The seed-specific expression cassettes containing either the FaoAor FaoB genes were inserted into the plant transformation vectorspCGN1559 (see FIG. 7) and pSBS2004. pCGN1559 contains CaMV 35S promoterdriving expression of the nptll gene (which confers resistance to theantibiotic, kanamycin) while pSBS2004 contains a parsley ubiquitinpromoter driving the PAT gene which confers resistance tophosphinothricine. Plasmids, pCGmf1P2S, pCGmf2P1S and PCGmf1A2P containboth FaoA and FaoB in the same binary vector (see FIG. 7).

[0139] Seed-Specific Arabidopsis-Reductase Construct

[0140] A plasmid pTRCN c.v. phaB was used as a template in anamplification reaction to obtain a 790 bp fragment encoding theacetoacyl CoA reductase from Chromatium vinosum. The PCR fragment wascloned into pBluescript and sequence analysis confirmed identity to theoriginal bacterial gene. The consumption of NADH measured at 340 nm inthe presence of acetoacetyl CoA showed that the activity of the geneproduct of the amplified fragment pTRCNRBSH-Rd108 gave similar activity,within the error of the assay, as the starting construct pTRCN c.v.phaB. The reductase fragment was cloned into pSBS2038 under the controlof the Arabidopsis oleosin promoter to obtain plasmid pM15006 shownbelow.

[0141] The seed-specific cassette for the expression of the reductasegene was cloned into the binary vector pCGN1559 to create plasmidpCGMI5006 for transformation into B. napus (see FIG. 7). Construct nameActivity (U/mg) pTRCNRBSH-Rd108 3.79 +/− 0.29 pTRCN C.v. phaB 3.40 +/−0.46 pTRCNRBSH 0.19 +/− 0.01

[0142] Seed-Specific and Constitutive Synthase Constructs

[0143] Similarly, the plasmid PMSXPB4C5Cat containing a fragmentencoding a hybrid Pseudomonas oleovorans/Zoogloea ramigera synthase wasused as a template to amplify a 1.79 kb fragment. The PCR fragment wascloned into pUC19 and sequenced. Functional analysis was performed atMetabolix Inc. by transforming the amplified fragment into an E. colistrain already expressing reductase and thiolase genes. This was grownin LB/glucose medium and was shown to make PHA. GC analysis of the wholeE. coli cell pellet showed the presence of PHA whereas a control strainwithout the amplified fragment did not. The amplified fragment wasinserted into the seed-specific promoter-terminator cassette pSBS2038resulting in plasmid pMI5038 as shown.

[0144] For the expression of the synthase gene in a constitutive manner,the amplified fragment was cloned into the plasmid pSBS2028 containingthe parsley ubiquitin promoter-terninator regulatory sequences alsoshown above. The Arabidopsis oleosin promoter-synthase and ubiquitinpromoter-synthase genes were subsequently cloned into the binary vectorpCGN1559 to generate plasmids pCGM15038 and pCGMI5028 respectively (seeFIG. 7) for transformation. Plasmid pM15034 contains both synthase andreductase coding sequences under the regulatory control of ubiquitin andoleosin promoters respectively.

[0145] FaoA Fusion to GUS Reporter Construct

[0146] To demonstrate that the FaoA and FaoB genes are transcribed andtranslated in a plant, a translational fusion with the E. colibetaglucuronidase (GUS) gene was made. The full length amplified FaoA(mf1) gene was fused in frame to GUS and the resulting fragment wasinserted into the expression cassette pSBS2038 which contained theArabidopsis oleosin regulatory sequences.

[0147] The final plasmid pGUSmf138 was used in biolistics experiments.To establish whether the FaoA gene would accumulate as a fusion proteinin plants, the chimaeric Arabidopsis-FaoA fragment was cloned into thebinary vector pCGN1559 and the resulting plasmid, pCGmfG138 was used totransform Brassica napus.

[0148] Plant Transformation

[0149] Agrobacterium-based binary vectors were used to transformcotyledons of 4 to 5 day old seedlings of Brassica napus cv. Westar.Table 3 below shows the various constructs used for transformation andthe number of transformed plants generated. Each construct comprises aparticular plant regulatory sequence and the bacterial coding sequenceswithin the binary vector pCGN1559. The number of transforned plants areindicated. Maps of the various constructs are also indicated in FIG. 7.Surviving transgenic plants of pha-FaoA were all sterile and unable toset seeds. Six out of sixteen transgenic plants from thepha-FaoA/soy-FaoB construct and two of soy-FaoA/pha-FaoB plants werealso sterile. TABLE 3 Transformation Constructs & Number of TransformedPlants Description Number of Construct name (promoter-bacterial gene)transformed plants pCGmf124 pha-FaoA 33 pCGmf125 soy-FaoA 18 pCGmf138Ara-FaoA 6 pCGmf224 pha-FaoB 39 pCGmf225 soy-FaoB 34 pCGmf1P2Spha-FaoA-soy-FaoB 16 pCGmf2P1S soy-FaoA-pha-FaoB 9 pCGmf1A2PAra-FaoA-pha-FaoB 9 PCGmfG138 Ara-FaoA-GUS 5 PCGMI5006 Ara-Red 10PCGMI5028 ubiq-Syn 10 PCGMI5038 Ara-Syn 6 PCGMI5034 ubiq-Syn-Ara-Red 2

EXAMPLE 10

[0150] Analysis of Transgenic Plants

[0151] All the transgenic plants showed nonnal development exceptpha-FaoA plants which were found to exhibit morphological changes. Theplants were sterile and therefore unable to set seed. They showedvigorous growth and produced more biomass. Characterization of thetransforming DNA by Southern blot showed that the FaoA gene had stablyintegrated into plant genome. Transient expression studies using a GUSreporter gene fused to FaoA demonstrated that the FaoA gene can betranscribed and translated in plants. Coexpression of both FaoA and FaoBin embryos also suggests the formation of a more stable complex. This issupported by transient expression studies where GUS activity in aGUS-FaoA fusion increased more than two fold when FaoB is coexpressed.Expression of FaoB was evident by the presence of the transcript andpolypeptide in transgenic plants. The expression of FaoA in plants wasfurther demonstrated by the detection of the transcript and polypeptidein plants transformed with a construct containing the Arabidopsisoleosin promoter regulating the FaoA gene. Changes in fatty acidprofiles of total seed lipid content in addition to an alteration inmorphology is evidence of functional expression of FaoA in transgenicplants. Northern and Western blot analysis also demonstratedtranscription and translation of the reductase and synthase genes intransgenic plants.

[0152] Morphological Changes in FaoA- and FaoB-Expressing Plants

[0153] Expression of the FaoA transgene under the control of thephaseolin promoter caused unexpected morphological changes in thetransgenic plants. The plants developed normally until flowering wherethe FaoA-expressing plants were found to be male sterile. This suggeststhat the phaseolin promoter regulating the FaoA gene was active duringmale gametogenesis. It has been demonstrated that phaseolin promoter isactive during microsporogenesis in transgenic tobacco (van der Geest, etal., Plant Physiol. 109:1151-58 (1995)). The plants visibly showedvigorous growth with a bushy appearance and produced more biomass whencompared with plants transformed with either the binary vector alone(pCGN1559 control) or containing soy-FaoA, pha-FaoB or soy-FaoBconstructs. This altered morphology is presumably caused by reducedfertility as these plants were unable to set seed. It should be notedthat seven of the male sterile plants were successfully crossed withpollen from soy-FaoB transgenic plants. It is likely that the functionalover-expression of the FaoA gene product has caused an alteration in afundamental process required for the normal development of the plant.Transgenic plants carrying the pha-FaoA/soy-FaoB and soy-FaoA/pha-FaoBconstructs on the other hand showed normal growth. It is thereforehypothesized that the accumulation of detrimental substrates resultingfrom the overexpression of functional FaoA may be converted to benignmetabolites when active FaoB protein is present.

[0154] Analysis of the Transgene in the Plant Genome

[0155] Successful gene transfer was confirmed by Southern blot analysisof total genomic isolated from leaves of the transgenic plants that hadbeen digested with Pvu II restriction enzyme. The enzyme cuts oncewithin the FaoA gene, the nptII gene and outside of the promotersequence. Hybridization analysis using a radiolabelled FaoAB gene probedemonstrated the stable integration of the FaoA gene. Intransgenicpha-FaoA plants (No. 15 and 44), the probe hybridized to theunexpected 2.4 and 2.8 kb fragments. The DNA containing soy-FaoAfragment in transgenic plants 69, 76, and 85 also appears to haveinserted stably, generating 1.4 and 2.2 kb fragments. The hybridizationpattern observed in plant number 82 seems to indicate that in thistransformant, the DNA had integrated into more than one site. Intransgenic plant 67, there appears to have been a rearrangement of theinserting DNA. Hybridization analysis of transgenic pha-FaoB plants (111and 121) also showed stable integration of the sequence. Theautoradiogram shows hybridization of the 32p_ labelled FaoAB gene probeto the expected 2.1 and 2.3 kb fragments. In transgenic plants thatharbor both FaoA and FaoB genes under the control of phaseolin andsoybean regulatory sequences, three hybridizing fragments (0.9, 2.8, and3.9 kb forpha-FaoA/soy-FaoB, and 2.1, 2.2, and 3.2 kbforsoy-FaoA/pha-FaoB plants) were expected with the probe. Thehybridizing bands in transgenic plant numbers 202 (pha-FaoA/soy/FaoB)and 252 (soy-FaoA/pha-FaoB) correctly indicated the expected DNA sizefragments. The probe shows some nonspecific hybridization at thestringency used, as some hybridization is also seen in the control(plant transformed with pCGN1559).

[0156] Analysis of MRNA and Protein Accumulation

[0157] Developing transgenic seeds were harvested at various stages andanalyzed for the expression of the bacterial FaoA and FaoB genes usingNorthern and Western analysis. Northern blot analysis was performed on30 μg of total seed RNA using radiolabelled DNA fragments representingthe coding sequence of the bacterial genes. For immunodetection,extracts of total seed protein were size-fractionated on 10-12%polyacrylamide-SDS gels and transferred to PVDF membrane. Antibodiesraised in rabbits against the bacterial FaoA or FaoB protein, and goatanti-rabbit IgG conjugated to horseradish peroxidase were used tovisualize the related polypeptides using chemiluminescence ECLimmunodetection.

[0158] Northern and Western blot analysis were performed on seedextracts from soy-FaoA transgenic plants which showed normal growth. Aweak signal of the related transcript was detected in one of fourtransgenic plants analyzed. The presence of the encoded polypeptide wastested by Western immunoblot analysis. In the transgenic plantsanalyzed, the anti-FaoA antibody did not detect the polypeptide. Thepresence of mRNA transcript from seeds of pha-FaoB (Plant No. 101, 102,103, 111, and 121) was also analyzed by Northern hybridization. Anoleosin probe was used as an internal standard to hybridize to the blotbefore it was partially stripped and reprobed with the radiolabelledFaoAB gene fragment. Hybridizing transcripts of expected size weredetected in five transgenic plants and absent from control plant as wellas FaoA-expressing (No. 22 and 77) plants. Immunoblot analysis ofFaoB-related polypeptide in plants showed that in the crude proteinextract of a mf111 plant, the anti-FaoB antibody cross-reacted with apolypeptide of approximately 43 kD similar in molecular weight to theFaoB standard. The extra non-specific hybridizing band in all samplesmay represent seed oilbody protein. No polypeptide was detected inone-fifth of plants analyzed from the same transgenic line. In addition,the anti-FaoB antibody did not bind to related polypeptide in samplestested from the soy-FaoB plants. It is likely that the related-FaoBpolypeptide is unable to accumulate as the protein is normallystabilized in vivo by the presence of the FaoA protein as demonstratedin bacterial systems. In some transgenic seeds of soy-FaoA/pha-FaoBanalyzed, hybridizing transcripts were detected for FaoB but not FaoA.However, the related polypeptide could not be detected by Western blotanalysis.

[0159] GC Analysis

[0160] Although the expression of the soy-FaoA fragment could not bedetected by Northern and Western blot analysis, the morphologicalalteration of transgenic plants resulting from pha-FaoA expression wasindirect evidence for the functional expression of an FaoA gene product.Since FaoA is a key component in oxidation of fatty acids, a profile oftotal seed lipid from soy-FaoA and control transgenic plants wasanalyzed. Fatty acid methyl esters were prepared according to Kunst, etal., “Fatty acid elongation in developing seeds of Arabidopsisthaliana.” Plant Physiol. Biochem. 30:425-34 (1992) and analyzed by gaschromatography. The chromatogram shows an enhanced peak of a lowmolecular weight fatty acid (arrowed) which was absent in the controltransgenic plant. This enhanced peak was also observed in a planttransformed with the pha-FaoA/soy-FaoB construct. The same peak wasobserved in eleven other transgenic plants including a male-sterilephas-FaoA plant fertilized with pollen from soy-FaoA plant. The resultssupport the conclusion that the FaoA polypeptide is functional andperturbs an essential metabolic process. A GC-MS analysis identified thepeak as pentanoic acid which would be an unusual cleavage product of afunctional FaoA.

[0161] Transient Assay of Expression of the FaoA and FaoB Genes inEmbryos

[0162] It is clear from the analysis presented that the pha-FaoBtranscript accumulates and is translated into its polypeptide. However,in an effort to demonstrate unequivocally that FaoA is indeedtranscribed and translated, a translational fusion with GUS at theC-terminus was made. The hypothesis was that if the reporter enzyme GUSaccumulates, then the FaoA gene must have been transcribed andtranslated. From the literature, it is known that FaoA and FaoB caninteract to form a stable complex in bacterial systems (Imamura, et al.,“Purification of the multienzyme complex for fatty acid oxidation fromPseudomonas fragi and reconstitution of the fatty acid oxidation system”J. Biochem. 107:184-89 (1990)). In order to test this hypothesis inplants, both FaoA and FaoB were expressed simultaneously in a transientmanner. An Ara-FaoA-GUS construct was used in this study in addition tothe pha-FaoB construct. The oilseed embryos used in this study were fromBrassica napus L. cv Topas and Linum usitatissimum (flax) cv. MacGregor.Microspore embryos were obtained from B. napus while zygotic embryoswere isolated from flax. Particle bombardment of embryos was essentiallyas described in Abenes, et al., “Transient expression and oil bodytargeting of an Arabidopsis oleosin-GUS reporter fusion protein in arange of oilseed embryos” Plant Cell Reports 17:1-7 (1997). Tables 4 and5 show GUS fluorimetric activities in the different fractions of embryoextracts. The GUS activity of Ara-GUS (pGN1.1) in microspore-derivedembryos was at least eight times the background (pSBS2105) while theactivity of Ara-FaoA-GUS (pmfG138) was more than double the backgroundactivity (Table 4). When embryos were co-bombarded with pmfG138(Ara-FaoA-GUS) and pmf224 (pha-FaoB) DNA in equal amounts, the specificactivity of GUS was observed to be more than three times that ofbackground activity. A comparison of the Ara-FaoA-GUS activity toAra-FaoA-GUS.pha-FaoB showed that the latter value was almost double theformer value when the background specific activity was subtracted. Itappears that the co-expression of both FaoA and FaoB contributed to theincrease in activity. This result was further confirmed when zygoticembryos were bombarded with the set of plasmids described in Table 4using microspore embryos. The data describes not only the use ofmicrospore and zygotic embryos to express FaoA and FaoB genes, but alsostresses the importance of the expression of these genes in differentplant species. TABLE 4 GUS activity levels in total homogenate ofmicrospore embryos from Brassica napus L. Cv. Topas. Construct GUSactivity Specific activity name Description (pmol MU/min) (activity/mgprt) pSBS2105 pBluescript- 30.5  7.78 based plasmid pGN1.1 Ara-GUS 256.553.1 pmfG138 Ara-FaoA-GUS 69 17.7 pmfG138: Ara-FaoA-GUS: 85 29.1 pmf224pha-FaoB

[0163] Table 5 shows the levels of GUS activity in oilbody andsupernatant (OS) and supernatant (SN) fractions. The recorded activityin the OS fraction was double the activity in SN fraction. It appearsthat some amount of GUS is also associated with oilbodies. This is mostlikely due to the hydrophobic nature of GUS and not the FaoA or FaoBprotein. In both fractions, there was an increase in GUS activity whenthe Ara-FaoA-GUS and pha-FaoB fragments were co-expressed in a ratio of1:1 over the Ara-FaoA-GUS. The activity increases further when the ratioof plasmid pmfG138:pmf224 DNA used was 1:3 (Table 5). The resultsobtained from transient assays of zygotic flax embryos confirmed theobservations noted in Table 4 when Brassica embryos were used. It isclear that the FaoA gene is transcribed and translated and that theproduct of FaoB gene expression increases the activity of the FaoA-GUSfusion protein. The effect of FaoB most likely occurs by forming acomplex with FaoA and stabilizing the FaoA domain within the FaoA-Gusfusion protein. TABLE 5 GUS activity levels in total homogenate (OS,oilbody fraction and supernatant) and supernatant (SN) of zygoticembryos from flax. GUS Specific activity activity Construct (pmol(activity/mg Fraction name Description MU/min) prt) OS pSBS2105pBluescript- 7.40 1.36 based pGN1.1 Ara-GUS 656.92 113.20 pmfG138Ara-FaoA-GUS 265.14 43.30 pmfG138: Ara-FaoA- 386.26 52.84 pmf224GUS:pha-FaoB (1:1) pmfG138: Ara-FaoA- 440.21 78.98 pmf224 GUS:pha-FaoB(1:3) SN pSBS2105 pBluescript- 2.55 0.47 based pGN1.1 Ara-GUS 373.8364.47 pmfG138 Ara-FaoA-GUS 132.38 21.62 pmfG138: Ara-FaoA- 174.54 23.88pmf224 GUS:pha-FaoB (1:1) pmfG138: Ara-FaoA- 214.07 38.41 pmf224GUS:pha-FaoB (1:3)

EXAMPLE 11

[0164] Comparison of Promoters.

[0165] Using the phaseolin promoter as a regulatory sequence to expressthe FaoA gene proved lethal to the normal development of transgenic B.napus plants, which indicates expression of a functional FoaA.Furthermore, the soybean oleosin promoter was comparatively weaker inexpressing either the FaoA or FaoB transgenes. In an effort to expressthe FaoA transgene in a seed-specific manner, a relatively strongArabidopsis oleosin promoter was used. An Ara-FaoA construct wasassembled in plasmid pCGNI 559 and used to transform B. napus. Planttransformation was also initiated with the Ara-Red, Ara-Syn, andubiq-Syn constructs. The following analyses were conducted on some ofthe transgenic plants obtained.

[0166] Analysis of Integration of Chimeric Arabidopsis-FaoA DNA Fragment

[0167] A Southern blot was prepared from 30 μg of total plant genomicDNA digested with EcoR V. A radiolabelled coding sequence of the FaoAgene was used as a probe for hybridization. There was successfulintegration of the transgene into the plant genome in four of thesamples analyzed. The number of hybridizing fragments indicate one ortwo copies of the insertion within the plant genome. The probe did nothybridize to DNA from the control plant.

[0168] Analysis of Expression of the FaoA Transgene in B. napus

[0169] A Northern blot was prepared using 30 μg of total RNA extractedfrom transgenic seeds. A ³²P-labelled FaoA gene probe hybridized to therelated transcript of expected size in transgenic plants. Nohybridization was observed with RNA from the control plant. Forimmunodetection, total seed protein extracts were size-fractionated on10-12% polyacrylamide-SDS gels and transferred to PVDF membrane.Antibodies raised in rabbits against the bacterial FaoA, and FaoBprotein, and goat anti-rabbit IgG conjugated to alkaline phosphatase(AP), were used to visualize the related polypeptides by using NBT andBCIP as AP substrates. Immunoblotting of 300 μg of total seed proteinprepared from Ara-FaoA plants with an anti-FaoAB antibody showed thatthe FaoA gene was both transcribed and translated in B. napus. Thecross-reacting polypeptide from the protein extract had the samemolecular mass as the purified FaoA protein standard. No immunoreactionto a related polypeptide was detected in control plant extracts.Nonspecific hybridization of the antibody with seed storage proteins,present in high amounts in the later development stages of B. napus,account for the signal seen in all plants. The results clearlydemonstrate that the Pseudomonas putida FaoA gene is expressed in B.napus plants when the Arabidopsis oleosin promoter is used to regulateexpression.

[0170] Analysis of FaoA/FaoB-Expressing Transgenic Plants

[0171] Northern and Western blot analysis were also performed ontransgenic seeds from plants transformed with a construct containingboth FaoA and FaoB genes on the same binary vector. FaoA and FaoB wereunder the regulation of Arabidopsis oleosin and phaseolin promotersrespectively. Autoradiography shows the respective transcripts in bothgenes from plant number 507; however, no transcripts were detected incontrol and three other plants. In Western blot analysis of total seedprotein from plant number 504 and 507, only the FaoA polypeptide couldbe detected. Although, a transcript could be detected with the FaoBprobe, the related polypeptide was not detected in the Western analysisusing the anti-FaoAB antibody.

[0172] Analysis of Expression of the Reductase Transgene in B. napus

[0173] In order to determine if the reductase gene was expressed intransgenic plants, the cloned reductase coding sequence wasradiolabelled and used as a probe in Northern blot hybridization.Autoradiography shows that the probe did not hybridize to RNA from thecontrol plant. In contrast, mRNA from two of the three transgenic plantsanalyzed, hybridized to the reductase gene probe. To examine thetranslational product resulting from the transcription of the reductasegene, a Western blot was prepared with 300 μg of protein extract in allfour samples analyzed and the polypeptide co-migrated with the purifiedbacterial reductase protein standard. There was no immunodetection of arelated polypeptide in the control plants. The extra nonspecifichybridizing band may represent accumulating oilbody protein in matureseeds. This result suggests that the bacterial reductase gene istranscribed and translated in B. napus plant.

[0174] Expression of the Synthase Gene in Transgenic Plants

[0175] To examine the expression of the hybrid synthase in transgenicplants in a constitutive as well as seed-specific manner, total RNA wasisolated from seeds. Thirty micrograms of RNA blotted onto nylonmembrane was hybridized with a ³²P-labelled synthase gene. Relatedtranscripts from two of the three ubiq-syn transgenic plants showedcross-hybridization with the complementary probe while no signal wasobserved in the control plant. Although the gene was transcribed asrevealed by the Northern analysis, the related polypeptide could not bedetected by Western blot analysis of protein extracts from leaves aswell as seeds using the anti-synthase antibody. However, a similartranscript was detected on Northern blot of total RNA isolated fromAra-syn transgenic plants and the related polypeptide was immunodetectedwith the anti-synthase antibody. The related polypeptide co-migratedwith the purified synthase and showed the same degradation products. Inaddition, a low molecular weight cleavage product was observed in thetransgenic lines analyzed.

[0176] Synthesis of Polymer in Embryos

[0177] As previously demonstrated in this study, the β-oxidation enzymesFaoA and FaoB can be transcribed and translated in embryo cells. In anattempt to synthesize PHA via β-oxidation of fatty acids in a transientfashion, flax zygotic embryos were co-bombarded with the Ara-FaoA,pha-FaoB, Ara-Red, and Ara-Syn constructs. A further biolisticexperiment was performed on another set of embryos with either theAra-Syn or gold particles alone. Butanolysis of embryos using PHB asinternal standard in the solvents ethanol, methanol, chloroform ,andhexane was performed at Metabolix Inc. GC analysis of the chromatogramsfrom ethanol and methanol soluble fractions did not show any differencesbetween samples. However, in the chloroform and hexane solublefractions, enhanced peaks at about 16.5 min in samples 2 (Ara-Syn) and 3(Ara-FaoA, pha-FaoB, Ara-Red and Ara-Syn) were observed. The peaks werenot present in sample I which was bombarded with gold-coated particlealone. The GC analysis could not detect PHB which is extractable inchloroform in any of the samples. Some conclusions drawn from thisanalysis suggest that if there was PHB in the samples, it would be lessthan 0.3% of the total cell dry weight of the samples analyzed, because0.24 mg of PHB standard could be detected on the GC. Secondly, theunidentified peaks are chloroform and hexane extractable and a mediumchain-length polymer would be expected to be extractable in bothsolvents. GC-MS analysis can be performed to identify these compounds.It should be noted that these peaks could not be found in GC analysis ofinsoluble fractions or residual cell matter.

[0178] Modifications and variations of the present invention will beobvious to those of skill in the art from the foregoing detaileddescription. Such modifications and variations are intended to comewithin the scope of the following claims.

We claim:
 1. A method for manipulating the metabolism of a plant,comprising expressing heterologous genes encoding fatty acid oxidationenzymes in the cytosol or plastids other than the peroxisomes,glyoxisomes or mitochondria, of the plant.
 2. The method of claim 1wherein the fatty acid β-oxidation enzymes are expressed from genes fromselected from the group consisting of bacterial, yeast, fungal, plant,and mammalian genes.
 3. The method of claim 2 wherein the fatty acidoxidation enzymes are expressed from genes from bacteria selected fromthe group consisting of Escherichia, Pseudomonas, Alcaligenes, andCoryneform.
 4. The method claim 3 wherein the genes are Pseudomonasputida faoAB.
 5. The method of claim 1 further comprising expressinggenes encoding enzymes selected from the group consisting ofpolyhydroxyalkanoate synthases, acetoacetyl-CoA reductases,β-ketoacyl-CoA thiolases, and enoyl-CoA hydratases.
 6. A DNA constructfor use in a method of manipulating the metabolism of a plant cellcomprising, in phase, (a) a promoter region functional in a plant; (b) astructural DNA sequence encoding at least one fatty acid oxidationenzyme activity; and (c) a 3′ nontranslated region of a gene naturallyexpressed in a plant, wherein the nontranslated region encodes a signalsequence for polyadenylation of mRNA.
 7. The DNA construct of claim 6wherein the promoter is a seed specific promoter.
 8. The DNA constructof claim 7 wherein the seed specific promoter is selected from the groupconsisting of napin promoter, phaseolin promoter, oleosin promoter, 2Salbumin promoter, zein promoter, β-conglycinin promoter, acyl-carrierprotein promoter, and fatty acid desaturase promoter.
 9. The DNAconstruct of claim 6 wherein the promoter is a constitutive promoter.10. The DNA construct of claim 6 wherein the promoter is selected fromthe group consisting of CaMV 35S promoter, enhanced CaMV 35S promoter,and ubiquitin promoter.
 11. A method for enhancing the biologicalproduction of polyhydroxyalkanoates in a transgenic plant, comprisingexpressing genes encoding heterologous fatty acid oxidation enzymes incytosol or plastids other than the peroxisomes, glyoxisomes ormitochondria, of the plant.
 12. The method of claim 11 wherein thetransgenic plant is selected from the group consisting of Brassica,maize, soybean, cottonseed, sunflower, palm, coconut, safflower, peanut,mustards, flax, tobacco, and alfalfa.
 13. A transgenic plant or partthereof comprising heterologous genes encoding fatty acid oxidationenzymes in cytosol or plastids other than the peroxisomes, glyoxisomesor mitochondria of the plant.
 14. The plant or part thereof of claim 13wherein the fatty acid β-oxidation enzymes are expressed from genesselected from the group consisting of bacterial, yeast, fungal, plant,and mammalian.
 15. The plant or part thereof of claim 14 wherein thefatty acid oxidation enzymes are expressed from genes from bacteriaselected from the group consisting of Escherichia, Pseudomonas,Alcaligenes, and Coryneform.
 16. The plant or part thereof of claim 15wherein the genes are Pseudomonas putida faoAB.
 17. The plant or partthereof of claim 13 further comprising genes encoding enzymes selectedfrom the group consisting of polyhydroxyalkanoate synthases,acetoacetyl-CoA reductases, β-ketoacyl-CoA thiolases, and enoyl-CoAhydratases.
 18. The plant or part thereof of claim 13 wherein the plantis selected from the group consisting of Brassica, maize, soybean,cottonseed, sunflower, palm, coconut, safflower, peanut, mustards, flax,tobacco, and alfalfa.
 19. The plant or part thereof of claim 13comprising a DNA construct comprising, in phase, (a) a promoter regionfunctional in a plant; (b) a structural DNA sequence encoding at leastone fatty acid oxidation enzyme activity; and (c) a 3′ nontranslatedregion of a gene naturally expressed in a plant, wherein thenontranslated region encodes a signal sequence for polyadenylation ofmRNA.
 20. The plant or part thereof of claim 19 wherein the promoter isa seed specific promoter.
 21. The plant or part thereof of claim 20wherein the seed specific promoter is selected from the group consistingof napin promoter, phaseolin promoter, oleosin promoter, 2S albuminpromoter, zein promoter, β-conglycinin promoter, acyl-carrier proteinpromoter, and fatty acid desaturase promoter.
 22. The plant or partthereof of claim 19 wherein the promoter is a constitutive promoter. 23.The plant or part thereof of claim 19 wherein the promoter is selectedfrom the group consisting of CaMV 35S promoter, enhanced CaMV 35Spromoter, and ubiquitin promoter.
 24. A method of preventing orsuppressing seed production in a plant, comprising expressingheterologous genes encoding fatty acid oxidation enzymes in cytosol orplastids other than the peroxisomes, glyoxisomes or mitochondria, of theplant.